Methods of inhibiting an immune response with anti-human endokine alpha antibodies

ABSTRACT

The present invention concerns a novel member of the tumor necrosis factor (TNF) family of cytokines. In particular, isolated nucleic acid molecules are provided encoding the endokine alpha protein. Endokine alpha polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic and therapeutic methods concerning TNF family-related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/136,511, filed May 2, 2002 now U.S. Pat. No. 7,078,027, which is adivisional of U.S. application Ser. No. 09/513,584, filed Feb. 25, 2000(now U.S. Pat. No. 6,406,867, issued Jun. 18, 2002), which claims thebenefit of U.S. Provisional Applications Nos. 60/136,788, filed May 28,1999, and No. 60/122,099, filed Feb. 26, 1999; said Ser. No. 09/513,584is also a continuation-in-part of U.S. application Ser. No. 09/345,790,filed Jul. 1, 1999, (now U.S. Pat. No. 6,521,742, issued Feb. 18, 2003),which is a divisional of U.S. application Ser. No. 08/912,227, filedAug. 15, 1997, (now U.S. Pat. No. 5,998,171, issued Dec. 7, 1999); saidSer. No. 08/912,227 claims the benefit of U.S. application Ser. No.60/024,058, filed Aug. 16, 1996. Each of the above-identifiedapplications is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a novel member of the tumor necrosisfactor (TNF) family of cytokines. In particular, isolated nucleic acidmolecules are provided encoding the endokine alpha protein. Endokinealpha polypeptides are also provided, as are vectors, host cells andrecombinant methods for producing the same. Also provided are diagnosticand therapeutic methods concerning TNF family-related disorders.

2. Related Art

The cytokine known as tumor necrosis factor-α (TNFα; also termedcachectin) is a protein secreted primarily by monocytes and macrophagesin response to endotoxin or other stimuli as a soluble homotrimer of 17kD protein subunits (Smith, R. A. et al., J. Biol. Chem. 262:6951-6954(1987)). A membrane-bound 26 kD precursor form of TNF has also beendescribed (Kriegler, M. et al., Cell 53:45-53 (1988)).

Accumulating evidence indicates that TNF is a regulatory cytokine withpleiotropic biological activities. These activities include: inhibitionof lipoprotein lipase synthesis (“cachectin” activity) (Beutler, B. etal., Nature 316:552 (1985)), activation of polymorphonuclear leukocytes(Klebanoff, S. J. et al., J. Immunol. 136:4220 (1986); Perussia, B., etal., J. Immunol. 138:765 (1987)), inhibition of cell growth orstimulation of cell growth (Vilcek, J. et al., J. Exp. Med. 163:632(1986); Sugarman, B. J. et al., Science 230:943 (1985); Lachman, L. B.et al., J. Immunol. 138:2913 (1987)), cytotoxic action on certaintransformed cell types (Lachman, L. B. et al., supra; Darzynkiewicz, Z.et al., Canc. Res. 44:83 (1984)), antiviral activity (Kohase, M. et al.,Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986)),stimulation of bone resorption (Bertolini, D. R. et al., Nature 319:516(1986); Saklatvala, J., Nature 322:547 (1986)), stimulation ofcollagenase and prostaglandin E2 production (Dayer, J.-M. et al., J.Exp. Med. 162:2163 (1985)); and immunoregulatory actions, includingactivation of T cells (Yokota, S. et al., J. Immunol. 140:531 (1988)), Bcells (Kehrl, J. H. et al., J. Exp. Med. 166:786 (1987)), monocytes(Philip, R. et al., Nature 323:86 (1986)), thymocytes (Ranges, G. E. etal., J. Exp. Med. 167:1472 (1988)), and stimulation of the cell-surfaceexpression of major histocompatibility complex (MHC) class I and classII molecules (Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446(1986); Pujol-Borrel, R. et al., Nature 326:304 (1987)).

TNF is noted for its pro-inflammatory actions which result in tissueinjury, such as induction of procoagulant activity on vascularendothelial cells (Pober, J. S. et al., J. Immunol. 136:1680 (1986)),increased adherence of neutrophils and lymphocytes (Pober, J. S. et al.,J. Immunol. 138:3319 (1987)), and stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).

Recent evidence implicates TNF in the pathogenesis of many infections(Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders,neoplastic pathology, e.g., in cachexia accompanying some malignancies(Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologiesand graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med.166:1280 (1987)). The association of TNF with cancer and infectiouspathologies is often related to the host's catabolic state. A majorproblem in cancer patients is weight loss, usually associated withanorexia. The extensive wasting which results is known as “cachexia”(Kern, K. A. et al. J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexiaincludes progressive weight loss, anorexia, and persistent erosion ofbody mass in response to a malignant growth. The cachectic state is thusassociated with significant morbidity and is responsible for themajority of cancer mortality. A number of studies have suggested thatTNF is an important mediator of the cachexia in cancer, infectiouspathology, and in other catabolic states.

TNF is thought to play a central role in the pathophysiologicalconsequences of Gram-negative sepsis and endotoxic shock (Michie, H. R.et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., SecondVienna Shock Forum, p. 463-466 (1989); Simpson, S. Q. et al., Crit. CareClin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.Endotoxin is a potent monocyte/macrophage activator which stimulatesproduction and secretion of TNF (Kombluth, S. K. et al., J. Immunol.137:2585-2591 (1986)) and other cytokines. Because TNF could mimic manybiological effects of endotoxin, it was concluded to be a centralmediator responsible for the clinical manifestations ofendotoxin-related illness. TNF and other monocyte-derived cytokinesmediate the metabolic and neurohormonal responses to endotoxin (Michie,H. R. et al., N. Eng. J. Med. 318:1481-1486 (1988)). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, A. et al., Arch. Surg. 123:162-170(1988)). Elevated levels of circulating TNF have also been found inpatients suffering from Gram-negative sepsis (Waage, A. et al., Lancet1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p.715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17:489-497(1989); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990)).

Passive immunotherapy directed at neutralizing TNF may have a beneficialeffect in Gram-negative sepsis and endotoxemia, based on the increasedTNF production and elevated TNF levels in these pathology states, asdiscussed above.

Antibodies to a “modulator” material which was characterized ascachectin (later found to be identical to TNF) were disclosed by Ceramiet al. (EPO Patent Publication 0,212,489, Mar. 4, 1987). Such antibodieswere said to be useful in diagnostic immunoassays and in therapy ofshock in bacterial infections. Rubin et al. (EPO Patent Publication0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human TNF,the hybridomas secreting such antibodies, methods of producing suchantibodies, and the use of such antibodies in immunoassay of TNF. Yoneet al. (EPO Patent Publication 0,288,088, Oct. 26, 1988) disclosedanti-TNF antibodies, including mAbs, and their utility in immunoassaydiagnosis of pathologies, in particular Kawasaki's pathology andbacterial infection. The body fluids of patients with Kawasaki'spathology (infantile acute febrile mucocutaneous lymph node syndrome;Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T., Shonica (Pediatrics)26:935 (1985)) were said to contain elevated TNF levels which wererelated to progress of the pathology (Yone et al., supra).

Other investigators have described mAbs specific for recombinant humanTNF which had neutralizing activity in vitro (Liang, C-M. et al.Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, A. et al.,Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987);Bringman, T. S. et al., Hybridoma 6:489-507 (1987); Hirai, M. et al., J.Immunol. Meth. 96:57-62 (1987); Moller, A. et al. (Cytokine 2:162-169(1990)). Some of these mAbs were used to map epitopes of human TNF anddevelop enzyme immunoassays (Fendly et al., supra; Hirai et al., supra;Moller et al., supra) and to assist in the purification of recombinantTNF (Bringman et al., supra). However, these studies do not provide abasis for producing TNF neutralizing antibodies that can be used for invivo diagnostic or therapeutic uses in humans, due to immunogenicity,lack of specificity and/or pharmaceutical suitability.

Neutralizing antisera or mAbs to TNF have been shown in mammals otherthan man to abrogate adverse physiological changes and prevent deathafter lethal challenge in experimental endotoxemia and bacteremia. Thiseffect has been demonstrated, e.g., in rodent lethality assays and inprimate pathology model systems (Mathison, J. C. et al., J. Clin.Invest. 81:1925-1937 (1988); Beutler, B. et al., Science 229:869-871(1985); Tracey, K. J. et al., Nature 330:662-664 (1987); Shimamoto, Y.et al., Immunol. Lett. 17:311-318 (1988); Silva, A. T. et al., J.Infect. Dis. 162:421-427 (1990); Opal, S. M. et al., J. Infect. Dis.161:1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279-292(1990)).

To date, experience with anti-TNF mAb therapy in humans has been limitedbut shows beneficial therapeutic results, e.g., in arthritis and sepsis.See, e.g., Elliott, M. J. et al., Baillieres Clin. Rheumatol. 9:633-52(1995); Feldmann M, et al., Ann. N.Y. Acad. Sci. USA 766:272-8 (1995);van der Poll, T. et al., Shock 3:1-12 (1995); Wherry et al., Crit. Care.Med. 21:S436-40 (1993); Tracey K. J., et al., Crit. Care Med. 21:S415-22(1993).

Sequence analysis of cytokine receptors has defined several subfamiliesof membrane proteins (1) the Ig superfamily, (2) the hematopoietin(cytokine receptor superfamily and (3) the tumor necrosis factor(TNF)/nerve growth factor (NGF) receptor superfamily (for review of TNFsuperfamily see, Gruss and Dower, Blood 85(12):3378-3404 (1995) andAggarwal and Natarajan, Eur. Cytokine Netw., 7(2):93-124 (1996)). TheTNF/NGF receptor superfamily contains at least 10 different proteins.Gruss and Dower, supra. Ligands for these receptors have been identifiedand belong to at least two cytokine superfamilies. Gruss and Dower,supra.

Accordingly, there is a need to provide cytokines similar to TNF thatare involved in pathological conditions. Such novel cytokines could beused to make novel antibodies or other antagonists that bind theseTNF-like cytokines for therapy of TNF-like disorders.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a cytokine that is similar to TNFand is believed to have similar biological effects and activities. Thiscytokine is named endokine alpha, and includes endokine alphapolypeptides having at least a portion of the amino acid sequence inFIG. 1 (SEQ ID NO:2) or an amino acid sequence encoded by the cDNA clonedeposited in a bacterial host as ATCC Deposit Number 97640 on Jun. 27,1996. The nucleotide sequence, which was determined by sequencing thedeposited endokine alpha cDNA clone, contains an open reading frameencoding a polypeptide of about 169 amino acid residues including anN-terminal methionine, an intracellular domain of about 17 amino acidresidues, a transmembrane domain of about 26 amino acids, anextracellular domain of about 126 amino acids, and a deduced molecularweight for the complete protein of about 19 kDa.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the endokine alpha polypeptide having the complete amino acidsequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the endokinealpha polypeptide having the complete amino acid sequence in SEQ ID NO:2but minus the N-terminal methionine residue; (c) a nucleotide sequenceencoding the endokine alpha polypeptide having the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97640;and (d) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b) or (c) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise or, alternatively, consist of, a polynucleotidehaving a nucleotide sequence at least 80%, 85%, 90%, 92%, or 95%identical, and more preferably at least 96%, 97%, 98% or 99% identical,to any of the nucleotide sequences in (a), (b), (c), or (d), above, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide in (a), (b), (c), or (d), above. This polynucleotidewhich hybridizes does not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues. An additional nucleic acidembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a endokine alpha polypeptide having an aminoacid sequence in (a), (b), (c), or (d), above.

The invention is further directed to nucleic acid fragments of thenucleic acid molecules described herein. Preferred nucleic acidfragments include nucleic acid molecules which encode: a polypeptidecomprising the endokine alpha intracellular domain (amino acid residuesfrom about 1 to about 17 in FIG. 1 (SEQ ID NO:2)); a polypeptidecomprising the endokine alpha transmembrane domain (amino acid residuesfrom about 18 to about 43 in FIG. 1 (SEQ ID NO:2)); and a polypeptidecomprising the endokine alpha extracellular domain (amino acid residuesfrom about 44 to about 169 in FIG. 1 (SEQ ID NO:2)).

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofendokine alpha polypeptides or peptides by recombinant techniques.

The invention further provides an isolated endokine alpha polypeptidehaving an amino acid sequence selected from the group consisting of: (a)the complete 169 amino acid sequence in SEQ ID NO:2; (b) the complete169 amino acid sequence in SEQ ID NO:2 but minus the N-terminalmethionine residue; (c) the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97640; and (d) the amino acidsequence of an epitope-bearing portion of any one of the polypeptides of(a), (b), or (c). The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80%, 85%, 90%, 92%,or 95% identical, more preferably at least 96%, 97%, 98% or 99%identical to those above.

Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a endokine alpha polypeptide of the inventioninclude portions of such polypeptides with at least six or seven,preferably at least nine, and more preferably at least about 30 aminoacids to about 50 amino acids, although epitope-bearing polypeptides ofany length up to and including the entire amino acid sequence of apolypeptide of the invention described above also are included in theinvention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to an endokine alpha polypeptide having an amino acidsequence described in (a), (b), (c), or (d) above.

Preferred polypeptide fragments according to the present inventioninclude a polypeptide comprising: the endokine alpha intracellulardomain, the endokine alpha transmembrane domain, and the endokine alphaextracellular domain.

The invention further provides methods for isolating antibodies thatbind specifically to an endokine alpha polypeptide having an amino acidsequence as described above. Such antibodies may be usefuldiagnostically or therapeutically as antagonists in the treatment ofendokine alpha- and/or TNF-related disorders. The invention alsoprovides a diagnostic method for determining the presence of aTNF-related disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ IDNO:2) sequences of the endokine alpha protein. Amino acids 1 to 17represent the intracellular domain, amino acids 18 to 43 thetransmembrane domain (the underlined sequence), and amino acids 44 to169 the extracellular domain (the remaining sequence).

FIG. 2 shows the regions of similarity between the amino acid sequencesof the endokine alpha protein (SEQ ID NO:2), tissue necrosis factor α(TNF-α) (SEQ ID NO:3), and TNF-β (SEQ ID NO:4). The J. Hein method wasused with PAM 250 residue weight table. Shading with solid blackindicates residues that match consensus exactly.

FIG. 3 provides an analysis of the endokine alpha amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 44-54, 57-68, 69-78, 94-105, 108-132 and 148-158 inFIG. 1 correspond to the shown highly antigenic regions of the endokinealpha protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding an endokine alpha protein having anamino acid sequence shown in FIG. 1 (SEQ ID NO:2), which was determinedby sequencing a cloned cDNA. Endokine alpha is a novel member of thetumor necrosis factor (TNF) ligand family and shares sequence homologywith human TNFα and related TNF family members (FIG. 2). The nucleotidesequence shown in FIG. 1 (SEQ ID NO:1) was obtained by sequencing a cDNAclone, which was deposited on Jun. 27, 1996, at the American TypeCulture Collection, Patent Depository, 10801 University Boulevard,Manassas, Va. 20110-2209, and given accession number 97640. Thedeposited clone is contained in the pBluescript SK(−) plasmid(Stratagene, LaJolla, Calif.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.99% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the expected amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence in is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofFIG. 1 (SEQ ID NO:1) set forth using deoxyribonucleotide abbreviationsis intended to indicate an RNA molecule having a sequence in which eachdeoxynucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxynucleotide T hasbeen replaced by a ribonucleotide U.

Using the information provided herein, such as the nucleotide sequencein FIG. 1, a nucleic acid molecule of the present invention encoding anendokine alpha polypeptide can be obtained using standard cloning andscreening procedures, such as those for cloning cDNAs using mRNA asstarting material. Illustrative of the invention, the nucleic acidmolecule described in FIG. 1 (SEQ ID NO:1) was discovered in a cDNAlibrary derived from human brain striatum. Expressed sequence tagscorresponding to a portion of the endokine alpha cDNA were also found inseveral endothelial libraries and a fetal liver library.

The endokine alpha gene contains an open reading frame encoding aprotein of about 169 amino acid residues, an intracellular domain ofabout 17 amino acids (amino acid residues from about 1 to about 17 inFIG. 1 (SEQ ID NO:2)), a transmembrane domain of about 26 amino acids(amino acid residues from about 18 to about 43 in FIG. 1 (SEQ ID NO:2)),an extracellular domain of about 126 amino acids (amino acid residuesfrom about 44 to about 169 in FIG. 1 (SEQ ID NO:2)); and a deducedmolecular weight of about 19 kDa. The endokine alpha protein shown inFIG. 1 (SEQ ID NO:2) is about 30% similar and about 22% identical tohuman TNF-α, which can be accessed on GenBank as Accession No. U42764.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above the actual endokine alpha polypeptideencoded by the deposited cDNA comprises about 169 amino acids, but canbe anywhere in the range of about 154-184 amino acids. It will also beappreciated by reasonable persons of skill in the art that, depending onthe criteria used, the exact ‘address’ of the above-described endokinealpha protein domains may differ. Thus, for example, the exact locationof the endokine alpha intracellular, transmembrane and extracellulardomains shown in FIG. 1 (SEQ ID NO:2) may vary slightly (e.g., the exactaddress may differ by about 1 to about 5 residues compared to that shownin FIG. 1) depending on the criteria used to define the domain.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA can be the coding strand, also known as the sensestrand, or it can be the non-coding strand, also referred to as theanti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

However, a nucleic acid contained in a clone that is a member of alibrary (e.g., a genomic or cDNA library) that has not been isolatedfrom other members of the library (e.g., in the form of a homogeneoussolution containing the clone and other members of the library) or achromosome isolated or removed from a cell or a cell lysate (e.g., a“chromosome spread,” as in a karyotype), is not “isolated” for thepurposes of the invention. As discussed further herein, isolated nucleicacid molecules according to the present invention may be producednaturally, recombinantly, or synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising the open reading frame (ORF) shown in FIG. 1 (SEQID NO:1) and further include nucleic acid molecules substantiallydifferent than all or part of the ORF sequence shown in FIG. 1 (SEQ IDNO:1) but which, due to the degeneracy of the genetic code, still encodethe endokine alpha protein or a fragment thereof. Of course, the geneticcode is well known in the art. Thus, it would be routine for one skilledin the art to generate the degenerate variants described above.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the endokine alpha polypeptide having an amino acidsequence encoded by the cDNA of the clone deposited as ATCC Deposit No.97640 on Jun. 27, 1996. The invention further provides an isolatednucleic acid molecule having the nucleotide sequence shown in FIG. 1(SEQ ID NO:1) or the nucleotide sequence of the endokine alpha cDNAcontained in the above-described deposited clone, or a nucleic acidmolecule having a sequence complementary to one of the above sequences.Such isolated molecules, particularly DNA molecules, are useful asprobes for gene mapping by in situ hybridization with chromosomes andfor detecting expression of the endokine alpha gene in human tissue, forinstance, by Northern blot analysis. As described in detail below,detecting altered endokine alpha gene expression in certain tissues orbodily fluids is indicative of certain disorders.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA or the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 nt inlength are also useful according to the present invention as arefragments corresponding to most, if not all, of the nucleotide sequenceof the deposited cDNA or as shown in FIG. 1 (SEQ ID NO:1). In thiscontext, “about” includes the particularly recited value and valueslarger or smaller by several (5, 4, 3, 2 or 1) nucleotides. By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQID NO:1). Since the gene has been deposited and the nucleotide sequenceshown in FIG. 1 (SEQ ID NO 1) is provided, generating such DNA fragmentswould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication could easily be used togenerate fragments of various sizes. Alternatively, such fragments couldbe generated synthetically.

In addition, the present inventors have also identified the followingrelated cDNA clone: HEMCG04R (SEQ ID NO:11), which, by BLAST analysishas 94% identity to nucleotides 26 to 482 of SEQ ID NO:1.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising or,alternatively, consisting of, the endokine alpha intracellular domain(amino acid residues from about 1 to about 17 in FIG. 1 (SEQ ID NO 2),or as encoded by the cDNA clone contained in ATCC Deposit No. 97640); apolypeptide comprising or, alternatively, consisting of, the endokinealpha transmembrane domain (amino acid residues from about 18 to about43 in FIG. 1 (SEQ ID NO 2), or as encoded by the cDNA clone contained inATCC Deposit No. 97640); and a polypeptide comprising or, alternatively,consisting of, the endokine alpha extracellular domain (amino acidresidues from about 44 to about 169 in FIG. 1 (SEQ ID NO:2), or asencoded by the cDNA clone contained in ATCC Deposit No. 97640).

Further preferred nucleic acid fragments of the present inventioninclude nucleic acid molecules encoding epitope-bearing portions of theendokine alpha protein. In particular, such nucleic acid fragments ofthe present invention include nucleic acid molecules encoding apolypeptide comprising or, alternatively, consisting of one, two, threeor more of any of the following amino acid sequences and polynucleotidesencoding these polypeptides: amino acid residues from about 44 to about158 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 44 to about54 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 57 to about68 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 69 to about78 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 94 to about105 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 108 to about132 in FIG. 1 (SEQ ID NO:2); and amino acid residues from about 148 toabout 158 in FIG. 1 (SEQ ID NO:2). The inventors have determined thatthe above polypeptide fragments are antigenic regions of the endokinealpha protein. Methods for determining other such epitope-bearingportions of the endokine alpha protein are described in detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, thecomplement of an endokine alpha polynucleotide fragment describedherein, or the cDNA clone contained in ATCC Deposit 97640 made on Jun.27, 1996. By “stringent hybridization conditions” is intended overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-500 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1). By a portion of apolynucleotide of “at least 20 nt in length,” for example, is intended20 or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide, (e.g., the deposited cDNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1)). As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Sambrook, J. et al., eds., Molecular Cloning, A LaboratoryManual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), the entire disclosure of which is herebyincorporated herein by reference.

Since an endokine alpha cDNA clone has been deposited and its nucleotidesequence is provided in FIG. 1 (SEQ ID NO:1), generating polynucleotideswhich hybridize to a portion of the endokine alpha cDNA molecule wouldbe routine to the skilled artisan. For example, restriction endonucleasecleavage or shearing by sonication of the endokine alpha cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the endokine alpha cDNAmolecule. Alternatively, the hybridizing polynucleotides of the presentinvention could be generated synthetically according to knowntechniques.

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the endokine alpha cDNA shownin FIG. 1 (SEQ ID NO:1)), or to a complementary stretch of T (or U)resides, would not be included in a polynucleotide of the invention usedto hybridize to a portion of a nucleic acid of the invention, since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention thatencode an endokine alpha protein may include, but are not limited to,those encoding the amino acid sequence of the polypeptide, by itself;the coding sequence for the polypeptide and additional sequences, suchas a pre-, or pro- or prepro-protein sequence; the coding sequence ofthe polypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to, introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, e.g., ribosome binding and stability of mRNA; an additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thesequence encoding the polypeptide can be fused to a marker sequence,such as a sequence encoding a peptide which facilitates purification ofthe fused polypeptide. In certain preferred embodiments of this aspectof the invention, the marker amino acid sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (Qiagen, Inc.), amongothers, many of which are publicly and/or commercially available. Asdescribed in Gentz et al, Proc. Natl. Acad. Sci. USA 86:821-824 (1989),for instance, hexa-histidine provides for convenient purification of thefusion protein. The “HA” tag is another peptide useful for purificationwhich corresponds to an epitope derived from the influenza hemagglutinin(HA) protein, which has been described by Wilson et al., Cell 37:767(1984). Other such fusion proteins include the endokine alpha proteinfused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the endokine alpha protein. Variants can occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism. Non-naturally occurring variants can beproduced, e.g., using art-known mutagenesis techniques.

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants can be altered in codingor non-coding regions or both. Alterations in the coding regions canproduce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the endokine alpha protein or portionsthereof. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are nucleic acid molecules encodingthe endokine alpha protein having the amino acid sequence shown in FIG.1 (SEQ ID NO:2) or the endokine alpha amino acid sequence encoded by thedeposited cDNA clone.

Further embodiments of the invention include isolated nucleic acidmolecules comprising or, alternatively, consisting of, a polynucleotidehaving a nucleotide sequence at least 80%, 85%, 90%, 92% or 95%identical, and more preferably at least 96%, 97%, 98%, or 99% identicalto (a) a nucleotide sequence encoding the polypeptide having the aminoacid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding thepolypeptide having the amino acid sequence in SEQ ID NO:2, but lackingthe N-terminal methionine; (c) a nucleotide sequence encoding thepolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97640; or (d) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), or (c).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding an endokinealpha polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theendokine alpha polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

The reference (query) sequence may be the entire endokine alpha encodingnucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or any endokine alphapolynucleotide fragment as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to,for instance, the nucleotide sequence shown in FIG. 1 or to thenucleotide sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the BESTFIT program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). BESTFIT uses the local homology algorithm of Smith andWaterman, Adv. Appl. Math. 2:482-489 (1981), to find the best segment ofhomology between two sequences. When using BESTFIT or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referencenucleotide sequence and that gaps in homology of up to 5% of the totalnumber of nucleotides in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatch/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to such nucleic acid molecules whichare at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical toa nucleic acid sequence described above irrespective of whether theyencode a polypeptide having endokine alpha protein activity. This isbecause, even where a particular nucleic acid molecule does not encode apolypeptide having endokine alpha activity, one of skill in the artwould still know how to use the nucleic acid molecule, for instance, asa hybridization probe or a polymerase chain reaction (PCR) primer. Usesof the nucleic acid molecules of the present invention that do notencode a polypeptide having endokine alpha activity include, inter alia,(1) isolating the endokine alpha gene or allelic variants thereof from acDNA library; (2) in situ hybridization (FISH) to metaphase chromosomalspreads to provide precise chromosomal location of the endokine alphagene as described in Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting endokine alpha mRNA expression in specifictissues.

Preferred, however, are such nucleic acid molecules having sequences atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence described above which do, in fact, encode apolypeptide having endokine alpha protein activity. By “a polypeptidehaving endokine alpha activity” is intended polypeptides exhibitingsimilar, but not necessarily identical, activity as compared to theendokine alpha protein as measured in a particular biological assay.Endokine alpha activity can be assayed according to known methods. Forexample, a cytotoxicity assay or cell proliferation assay can be usedwhere endokine alpha polypeptides are added to cells in culture and theeffect of the endokine on the cells is determined by measuring thedecrease or increase in cell numbers.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequencedescribed above will encode a polypeptide “having endokine alpha proteinactivity.” In fact, since degenerate variants all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving endokine alpha protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990), wherein the authors indicate that there are twomain approaches for studying the tolerance of an amino acid sequence tochange. The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U., et al., supra, and the references cited therein.

By “a polypeptide having endokine alpha functional activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the endokine alpha receptors of the present invention(either the full-length polypeptide, or the splice variants), asmeasured, for example, in a particular immunoassay or biological assay.For example, endokine alpha activity can be measured by determining theability of an endokine alpha polypeptide to bind an endokine alphaligand (e.g., TR11 (GITR, AITR)). Endokine alpha activity may also bemeasured by determining the ability of a polypeptide, such as cognateligand which is free or expressed on a cell surface, to stimulateproliferation, differentiation or activation, or to stimulate TNF-αproduction, and/or to inhibit IL-12 production in cells expressing thepolypeptide, for example, B cells, T cells and monocytes.

The present invention is further directed to fragments of the isolatednucleic acid molecules (i.e. polynucleotides) described herein. By afragment of an isolated nucleic acid molecule having, for example, thenucleotide sequence of the deposited cDNA (clone 97640), a nucleotidesequence encoding the polypeptide sequence encoded by the depositedcDNA, a nucleotide sequence encoding the polypeptide sequence depictedin FIG. 1 (SEQ ID NO:2), the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1), or the complementary strand thereto, is intended fragments atleast 15 nucleotides, and more preferably at least about 20 nucleotides,still more preferably at least 30 nucleotides, and even more preferably,at least about 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450,500, 550, or 600 nucleotides in length. In this context, “about”includes the particularly recited value and values larger or smaller byseveral (5, 4, 3, 2 or 1) nucleotides. These fragments have numeroususes which include, but are not limited to, diagnostic probes andprimers as discussed herein. Of course, larger fragments, such as thoseof 501-1500 nucleotides in length are also useful according to thepresent invention as are fragments corresponding to most, if not all, ofthe nucleotide sequences of the deposited cDNA (clone 97640) or as shownin FIG. 1 (SEQ ID NO:1). By a fragment at least 20 nucleotides inlength, for example, is intended fragments which include 20 or morecontiguous bases from, for example, the nucleotide sequence of thedeposited cDNA, or the nucleotide sequence as shown in FIG. 1 (SEQ IDNO:1).

Representative examples of endokine alpha polynucleotide fragments ofthe invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to 50, 51to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651to 700, 701 to 750, 751 to 800, 801 to 850, 851 to 900, 901 to 950, 951to 1000, 1001 to 1050, 1051 to 1100, 1101 to 1150, 1151 to 1200, 1201 to1250, 1251 to 1300, 1301 to 1350, 1351 to 1400, 1401 to 1450, 1451 to1500, 1501 to 1550, 1551 to 1600, 1601 to 1650, 1651 to 1700, 1701 to1750, 1751 to 1800, and/or 1801 to 1840 of SEQ ID NO:1, or thecomplementary strand thereto, or the cDNA contained in the depositedclone. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini.

In specific embodiments, the polynucleotide fragments of the inventioncomprise, or alternatively, consist of, a sequence from nucleotide 961to 1000, 1730 to 1770, 1770 to 1800, and/or 1800 to 1840, of SEQ IDNO:1, or the complementary strand thereto. Polynucleotides thathybridize to these polynucleotide fragments are also encompassed by theinvention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates an endokine alpha functional activity. Bya polypeptide demonstrating “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length endokine alpha polypeptide.Such functional activities include, but are not limited to, biologicalactivity (e.g., stimulation of B cell proliferation, differentiation oractivation; stimulation of T cell proliferation, differentiation oractivation; stimulation of TNF-α production in monocytes; and/orinhibition of IL-12 production in monocytes), antigenicity (ability tobind (or compete with an endokine alpha polypeptide for binding) to ananti-endokine alpha antibody), immunogenicity (ability to generateantibody which binds to a endokine alpha polypeptide), ability tomultimerize with native endokine alpha and ability to bind to a receptoror ligand for a endokine alpha polypeptide (e.g., TR11; see U.S. patentapplication Ser. No. 09/176,200).

The functional activity of endokine alpha polypeptides, and fragments,variants, derivatives, and analogs thereof, can be assayed by variousmethods. For example, in one embodiment where one is assaying for theability to bind or compete with full-length endokine alpha polypeptidefor binding to anti-endokine alpha antibody, various immunoassays knownin the art can be used, including, but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where an endokine alpha ligand is identified(e.g., TR11), or the ability of a polypeptide fragment, variant orderivative of the invention to multimerize is being evaluated, bindingcan be assayed, e.g., by means well-known in the art, such as, forexample, reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., Microbiol. Rev. 59:94-123 (1995). In another embodiment,physiological correlates (signal transduction) of endokine alpha bindingto its substrates can be assayed.

In addition, assays described herein (see Examples 5-8) and methodsotherwise known in the art may routinely be applied to measure theability of endokine alpha polypeptides and fragments, variants,derivatives and analogs thereof to elicit endokine alpha relatedbiological activity (e.g., stimulation of B cell proliferation,differentiation or activation; stimulation of T cell proliferation,differentiation or activation; stimulation of TNF-α production inmonocytes; and/or inhibition of IL-12 production in monocytes in vitroor in vivo). Other methods will be known to the skilled artisan and arewithin the scope of the invention.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of endokinealpha polypeptides or portions thereof by recombinant techniques.

Recombinant constructs may be introduced into host cells using wellknown techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids. See, e.g., Ausubel, infra; Sambrook, infra.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli: lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include bacterial cells,such as E. coli, Streptomyces and Salmonella typhimurium cells; fungalcells, such as yeast cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanomacells; and plant cells. Appropriate culture media and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. In a further example, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, as indicated, a region(s) also may be addedto the polypeptide to facilitate purification. Such regions may beremoved prior to final preparation of the polypeptide. The addition ofpeptide moieties to polypeptides to engender secretion or excretion, toimprove stability and to facilitate purification, among others, arefamiliar and routine techniques in the art. A preferred fusion proteincomprises a heterologous region from immunoglobulin that is useful tosolubilize receptors. For example, EP A 0,464,533 (also, Canadiancounterpart 2,045,869) discloses fusion proteins comprising variousportions of constant region of immunoglobin molecules together withanother human protein or part thereof. In many cases, the Fc part in thefusion protein is thoroughly advantageous for use in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP A 0,232,262). On the other hand, for some uses it wouldbe desirable to be able to delete the Fc part after the fusion proteinhas been expressed, detected and purified in the advantageous mannerdescribed. This is the case when Fc portion proves to be a hindrance touse in therapy and diagnosis, for example when the fusion protein is tobe used as antigen for immunizations. In drug discovery, for example,human proteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists (forexample, hIL-5). See, D. Bennett et al., Journal of MolecularRecognition 8:52-58(1995) and K. Johanson et al., The Journal ofBiological Chemisty 270(16):9459-9471 (1995).

The endokine alpha protein can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., endokine alpha coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with endokine alpha polynucleotides of theinvention, and which activates, alters, and/or amplifies endogenousendokine alpha polynucleotides. For example, techniques known in the artmay be used to operably associate heterologous control regions (e.g.,promoter and/or enhancer) and endogenous endokine alpha polynucleotidesequences via homologous recombination (see, e.g., U.S. Pat. No.5,641,670, issued Jun. 24, 1997; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

Endokine Alpha Polypeptides and Peptides

The invention further provides an isolated endokine alpha polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in FIG. 1 (SEQ ID NO:2), or a peptide or polypeptidecomprising a portion of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of at least two amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in recombinant host cellsare considered isolated for purposes of the invention as are native orrecombinant polypeptides and proteins which have been substantiallypurified by any suitable technique such as, for example, the one-stepmethod described in Smith and Johnson, Gene 67:31-40 (1988).

It will be recognized in the art that some amino acid sequence of theendokine alpha polypeptide can be varied without significant effect ofthe structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity. In general, itis possible to replace residues which form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of the endokine alphapolypeptide which show substantial endokine alpha polypeptide activityor which include regions of endokine alpha protein such as the proteinfragments discussed below. Such mutants include deletions, insertions,inversions, repeats, and type substitutions (for example, substitutingone hydrophilic residue for another, but not strongly hydrophilic forstrongly hydrophobic as a rule). Small changes or such “neutral” aminoacid substitutions will generally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the endokine alpha protein. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, theendokine alpha of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given endokinealpha polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3,depending on the objective.

Amino acids in the endokine alpha protein of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the endokine alpha polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include the polypeptidescomprising or, alternatively, consisting of: (a) the complete amino acidsequence as shown in FIG. 1 (SEQ ID NO:2); (b) the complete amino acidsequence as shown in FIG. 1 (SEQ ID NO:2), but minus the N-terminalmethionine residue; (c) the amino acid sequence of the endokine alphapolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97640; and (d) the amino acid sequence ofan epitope-bearing portion of any one of the polypeptides of (a), (b),or (c), as well as polypeptides which are at least 80%, 85%, 90%, 92% or95% identical, more preferably at least 96%, 97%, 98% or 99% identicalto a polypeptide described herein, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an endokine alphapolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid sequence of the endokine alphapolypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide comprises or,alternatively, consists of, a sequence at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98% or 99% identical to, for instance, the amino acid sequenceshown in FIG. 1 (SEQ ID NO:2) or to the amino acid sequence encoded bydeposited cDNA clone can be determined conventionally using knowncomputer programs such the BESTFIT program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The present inventors have discovered that the endokine alpha protein isa 169 residue protein exhibiting three main structural domains. Theintracellular domain was identified within residues from about 1 toabout 17 in FIG. 1 (SEQ ID NO:2). The transmembrane domain wasidentified within residues from about 18 to about 43 in FIG. 1 (SEQ IDNO:2). The extracellular domain was identified within residues fromabout 44 to about 169 in FIG. 1 (SEQ ID NO:2). Thus, the inventionfurther provides preferred endokine alpha protein fragments comprising apolypeptide selected from: the endokine alpha intracellular domain, thetransmembrane domain and the endokine alpha extracellular domain.

The extracellular domain of the endokine alpha protein can be combinedwith parts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These fusion proteins show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing the ligands than the monomeric extracellular domains alone(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).

Polypeptide fragments of the present invention include polypeptidescomprising or alternatively, consisting of, an amino acid sequencecontained in SEQ ID NO:2, encoded by the cDNA contained in the depositedclone, or encoded by nucleic acids which hybridize (e.g., understringent hybridization conditions) to the nucleotide sequence containedin the deposited clone, or shown in FIG. 1 (SEQ ID NO:1) or thecomplementary strand thereto. Protein fragments may be “free-standing,”or comprised within a larger polypeptide of which the fragment forms apart or region, most preferably as a single continuous region.Representative examples of polypeptide fragments of the invention,include, for example, fragments that comprise or alternatively, consistof from about amino acid residues: 1 to 50, 51 to 100, 101 to 150 and/or151 to 169 of SEQ ID NO:2. In this context, “about” includes theparticularly recited ranges and ranges larger or smaller, by several (5,4, 3, 2, or 1) amino acids, at either terminus or both termini.Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150 or 168 amino acids in length.Polynucleotides encoding these polypeptides are also encompassed by theinvention. Polynucleotides that hybridize to the complement of theseencoding polynucleotides are also encompassed by the invention, as arethe polypeptides encoded by these hybridizing polynucleotides.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of endokine alpha.Such fragments include amino acid residues that comprise alpha-helix andalpha-helix-forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of full-length endokine alpha (SEQ ID NO:2).Certain preferred regions are those set out in FIG. 3 and include, butare not limited to, regions of the aforementioned types identified byanalysis of the amino acid sequence depicted in FIG. 1 (SEQ ID NO:2),such preferred regions include; Garnier-Robson predicted alpha-regions,beta-regions, turn-regions, and coil-regions; Chou-Fasman predictedalpha-regions, beta-regions, turn-regions, and coil-regions;Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenbergalpha and beta amphipathic regions; Emini surface-forming regions; andJameson-Wolf high antigenic index regions, as predicted using thedefault parameters of these computer programs. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

The data representing the structural or functional attributes ofendokine alpha set forth in FIG. 3 and/or Table 2, as described above,was generated using the various modules and algorithms of the DNA*STARset on default parameters. In a preferred embodiment, the data presentedin columns VIII, IX, XIII, and XIV of Table 2 can be used to determineregions of endokine alpha which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table 2, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table 2). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table 2include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 3. Asset out in FIG. 3 and in Table 2, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

Amino and Carboxy Terminal Deletions. As mentioned above, even ifdeletion of one or more amino acids from the N-terminus of a proteinresults in modification or loss of one or more biological functions ofthe protein, other biological activities may still be retained. Thus,the ability of shortened endokine alpha Madonnas to induce and/or bindto antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that an endokine alpha mutein witha large number of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six endokine alpha amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the endokinealpha amino acid sequence shown in FIG. 1 (i.e., SEQ ID NO:2), up to theasparagine residue at position number 164 and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising the amino acid sequence of residues n-169 ofFIG. 1 (SEQ ID NO:2), where n is an integer in the range of 2 to 164,and 165 is the position of the first residue from the N-terminus of thecomplete endokine alpha polypeptide believed to be required for at leastimmunogenic activity of the endokine alpha polypeptide.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of: residues P-2to S-169; L-3 to S-169; S-4 to S-169; H-5 to S-169; S-6 to S-169; R-7 toS-169; T-8 to S-169; Q-9 to S-169; G-10 to S-169; A-11 to S-169; Q-12 toS-169; R-13 to S-169; S-14 to S-169; S-15 to S-169; W-16 to S-169; K-17to S-169; L-18 to S-169; W-19 to S-169; L-20 to S-169; F-21 to S-169;C-22 to S-169; S-23 to S-169; I-24 to S-169; V-25 to S-169; M-26 toS-169; L-27 to S-169; L-28 to S-169; F-29 to S-169; L-30 to S-169; C-31to S-169; S-32 to S-169; F-33 to S-169; S-34 to S-169; W-35 to S-169;L-36 to S-169; I-37 to S-169; F-38 to S-169; I-39 to S-169; F-40 toS-169; L-41 to S-169; Q-42 to S-169; L-43 to S-169; E-44 to S-169; T-45to S-169; A-46 to S-169; K-47 to S-169; E-48 to S-169; P-49 to S-169;C-50 to S-169; M-51 to S-169; A-52 to S-169; K-53 to S-169; F-54 toS-169; G-55 to S-169; P-56 to S-169; L-57 to S-169; P-58 to S-169; S-59to S-169; K-60 to S-169; W-61 to S-169; Q-62 to S-169; M-63 to S-169;A-64 to S-169; S-65 to S-169; S-66 to S-169; E-67 to S-169; P-68 toS-169; P-69 to S-169; C-70 to S-169; V-71 to S-169; N-72 to S-169; K-73to S-169; V-74 to S-169; S-75 to S-169; D-76 to S-169; W-77 to S-169;K-78 to S-169; L-79 to S-169; E-80 to S-169; I-81 to S-169; L-82 toS-169; Q-83 to S-169; N-84 to S-169; G-85 to S-169; L-86 to S-169; Y-87to S-169; L-88 to S-169; I-89 to S-169; Y-90 to S-169; G-91 to S-169;Q-92 to S-169; V-93 to S-169; A-94 to S-169; P-95 to S-169; N-96 toS-169; A-97 to S-169; N-98 to S-169; Y-99 to S-169; N-100 to S-169;D-101 to S-169; V-102 to S-169; A-103 to S-169; P-104 to S-169; F-105 toS-169; E-106 to S-169; V-107 to S-169; R-108 to S-169; L-109 to S-169;Y-110 to S-169; K-11 to S-169; N-112 to S-169; K-113 to S-169; D-114 toS-169; M-115 to S-169; I-116 to S-169; Q-117 to S-169; T-118 to S-169;L-119 to S-169; T-120 to S-169; N-121 to S-169; K-122 to S-169; S-123 toS-169; K-124 to S-169; I-125 to S-169; Q-126 to S-169; N-127 to S-169;V-128 to S-169; G-129 to S-169; G-130 to S-169; T-131 to S-169; Y-132 toS-169; E-133 to S-169; L-134 to S-169; H-135 to S-169; V-136 to S-169;G-137 to S-169; D-138 to S-169; T-139 to S-169; I-140 to S-169; D-141 toS-169; L-142 to S-169; I-143 to S-169; F-144 to S-169; N-145 to S-169;S-146 to S-169; E-147 to S-169; H-148 to S-169; Q-149 to S-169; V-150 toS-169; L-151 to S-169; K-152 to S-169; N-153 to S-169; N-154 to S-169;T-155 to S-169; Y-156 to S-169; W-157 to S-169; G-158 to S-169; I-159 toS-169; I-160 to S-169; L-161 to S-169; L-162 to S-169; A-163 to S-169;and N-164 to S-169 of the endokine alpha sequence shown in FIG. 1. Thepresent invention is also directed to nucleic acid molecules comprisingor, alternatively, consisting of, a polynucleotide sequence at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequences encoding the endokine alpha polypeptidesdescribed above. The present invention also encompasses the abovepolynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these polynucleotide sequences arealso encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened endokine alphamutein to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan endokine alpha mutein with a large number of deleted C-terminal aminoacid residues may retain some biological or immunogenic activities. Infact, peptides composed of as few as six endokine alpha amino acidresidues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the endokine alpha polypeptide shown in FIG. 1 (SEQ IDNO:2), up to the serine residue at position number 6, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino acid sequence ofresidues 1-m of FIG. 1 (i.e., SEQ ID NO:2), where m is an integer in therange of 6 to 169, and 6 is the position of the first residue from theC-terminus of the complete endokine alpha polypeptide believed to berequired for at least immunogenic activity of the endokine alphapolypeptide.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of: residues M-1to I-168; M-1 to F-167; M-1 to Q-166; M-1 to P-165; M-1 to N-164; M-1 toA-163; M-1 to L-162; M-1 to L-161; M-1 to I-160; M-1 to I-159; M-1 toG-158; M-1 to W-157; M-1 to Y-156; M-1 to T-155; M-1 to N-154; M-1 toN-153; M-1 to K-152; M-1 to L-151; M-1 to V-150; M-1 to Q-149; M-1 toH-148; M-1 to E-147; M-1 to S-146; M-1 to N-145; M-1 to F-144; M-1 toI-143; M-1 to L-142; M-1 to D-141; M-1 to I-140; M-1 to T-139; M-1 toD-138; M-1 to G-137; M-1 to V-136; M-1 to H-135; M-1 to L-134; M-1 toE-133; M-1 to Y-132; M-1 to T-131; M-1 to G-130; M-1 to G-129; M-1 toV-128; M-1 to N-127; M-1 to Q-126; M-1 to I-125; M-1 to K-124; M-1 toS-123; M-1 to K-122; M-1 to N-121; M-1 to T-120; M-1 to L-119; M-1 toT-118; M-1 to Q-117; M-1 to I-116; M-1 to M-115; M-1 to D-114; M-1 toK-113; M-1 to N-112; M-1 to K-111; M-1 to Y-110; M-1 to L-109; M-1 toR-108; M-1 to V-107; M-1 to E-106; M-1 to F-105; M-1 to P-104; M-1 toA-103; M-1 to V-102; M-1 to D-101; M-1 to N-100; M-1 to Y-99; M-1 toN-98; M-1 to A-97; M-1 to N-96; M-1 to P-95; M-1 to A-94; M-1 to V-93;M-1 to Q-92; M-1 to G-91; M-1 to Y-90; M-1 to I-89; M-1 to L-88; M-1 toY-87; M-1 to L-86; M-1 to G-85; M-1 to N-84; M-1 to Q-83; M-1 to L-82;M-1 to I-81; M-1 to E-80; M-1 to L-79; M-1 to K-78; M-1 to W-77; M-1 toD-76; M-1 to S-75; M-1 to V-74; M-1 to K-73; M-1 to N-72; M-1 to V-71;M-1 to C-70; M-1 to P-69; M-1 to P-68; M-1 to E-67; M-1 to S-66; M-1 toS-65; M-1 to A-64; M-1 to M-63; M-1 to Q-62; M-1 to W-61; M-1 to K-60;M-1 to S-59; M-1 to P-58; M-1 to L-57; M-1 to P-56; M-1 to G-55; M-1 toF-54; M-1 to K-53; M-1 to A-52; M-1 to M-51; M-1 to C-50; M-1 to P-49;M-1 to E-48; M-1 to K-47; M-1 to A-46; M-1 to T-45; M-1 to E-44; M-1 toL-43; M-1 to Q-42; M-1 to L-41; M-1 to F-40; M-1 to I-39; M-1 to F-38;M-1 to I-37; M-1 to L-36; M-1 to W-35; M-1 to S-34; M-1 to F-33; M-1 toS-32; M-1 to C-31; M-1 to L-30; M-1 to F-29; M-1 to L-28; M-1 to L-27;M-1 to M-26; M-1 to V-25; M-1 to I-24; M-1 to S-23; M-1 to C-22; M-1 toF-21; M-1 to L-20; M-1 to W-19; M-1 to L-18; M-1 to K-17; M-1 to W-16;M-1 to S-15; M-1 to S-14; M-1 to R-13; M-1 to Q-12; M-1 to A-11; M-1 toG-10; M-1 to Q-9; M-1 to T-8; M-1 to R-7; and M-1 to S-6 of the sequenceof the endokine alpha sequence shown in FIG. 1. The present invention isalso directed to nucleic acid molecules comprising or, alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencesencoding the endokine alpha polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesepolynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of an endokinealpha polypeptide, which may be described generally as having residuesn-m of FIG. 1 (i.e., SEQ ID NO:2), where n and m are integers asdescribed above.

The endokine alpha polypeptides of the invention may be in monomers ormultimers (i.e., dimers, trimers, tetramers and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe endokine alpha polypeptides of the invention, their preparation, andcompositions (preferably, pharmaceutical compositions) containing them.In specific embodiments, the polypeptides of the invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyendokine alpha polypeptides of the invention (including endokine alphafragments, variants, and fusion proteins, as described herein). Thesehomomers may contain endokine alpha polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only endokine alpha polypeptideshaving an identical amino acid sequence. In another specific embodiment,a homomer of the invention is a multimer containing endokine alphapolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing endokine alpha polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing endokine alphapolypeptides having identical or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous polypeptides (i.e., polypeptides of a different protein) inaddition to the endokine alpha fragments and endokine alpha polypeptidesof the invention. In a specific embodiment, the multimer of theinvention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the heteromeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the endokine alpha polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptide encoded by the clone97640). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in an endokine alpha fusionprotein. In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained ina endokine alpha-Fc fusion protein of the invention (as describedherein). In another specific example, covalent associations of fusionproteins of the invention are between heterologous polypeptide sequencefrom another TNF family ligand/receptor member that is capable offorming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication No. WO 98/49305,the contents of which are herein incorporated by reference in itsentirety).

In another embodiment, two or more endokine alpha polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple endokine alphapolypeptides separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer endokine alpha polypeptides of theinvention involves use of endokine alpha polypeptides fused to a leucinezipper or isoleucine zipper polypeptide sequence. Leucine zipper andisoleucine zipper domains are polypeptides that promote multimerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericendokine alpha proteins are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a soluble endokine alpha polypeptide fused to a peptide thatdimerizes or trimerizes in solution are expressed in suitable hostcells, and the resulting soluble multimeric endokine alpha is recoveredfrom the culture supernatant using techniques known in the art.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,Cell 73:431, 1993). Thus, trimeric endokine alpha may offer theadvantage of enhanced biological activity. Preferred leucine zippermoieties are those that preferentially form trimers. One example is aleucine zipper derived from lung surfactant protein D (SPD), asdescribed in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric endokine alpha.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained inFlag®-endokine alpha fusion proteins of the invention. In a furtherembodiment, associations proteins of the invention are associated byinteractions between a heterologous polypeptide sequence contained inFlag®-endokine alpha fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al, 1984, Nature 310:105-111). For example, apeptide corresponding to a fragment of the endokine alpha polypeptidesof the invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into theendokine alpha polynucleotide sequence. Non-classical amino acidsinclude, but are not limited to, to the D-isomers of the common aminoacids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid,Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses endokine alpha polypeptides which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques,including, but not limited, to specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofendokine alpha which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The polypeptides of the present invention have uses which include, butare not limited to, as sources for generating antibodies that bind thepolypeptides of the invention, and as molecular weight markers onSDS-PAGE gels or on molecular sieve gel filtration columns using methodswell known to those of skill in the art.

Protein Modification

In addition, proteins of the invention can be chemically synthesizedusing techniques known in the art (see, e.g., Creighton, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983),and Hunkapiller, M., et al., Nature 310:105-111 (1984)). For example, apeptide corresponding to a fragment of the endokine-alpha polypeptidesof the invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into theendokine-alpha polypeptide sequence. Non-classical amino acids include,but are not limited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, alpha-Abu, alpha-Ahx, 6-amino hexanoicacid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, alpha-alanine, fluoro-amino acids,designer amino acids such as alpha-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acids can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), andrestriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

The invention additionally, encompasses endokine-alpha polypeptideswhich are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofendokine alpha which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on the functionalor antigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a protein via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus may be accomplished by reductive alkylationwhich exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. Nos. 4,002,531; 5,349,052; WO 95/06058; and WO 98/32466, thedisclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Antibodies and Epitopes

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting endokine alpha proteinexpression as described below or as agonists and antagonists capable ofinhibiting endokine alpha protein function. Further, such polypeptidescan be used in the yeast two-hybrid system to “capture” endokine alphaprotein binding proteins which are also candidate agonist and antagonistaccording to the present invention. The yeast two hybrid system isdescribed in Fields and Song, Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1984).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G. et al., Science219:660-666 (1983). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer, soluble peptides, especially those containing proline residues,usually are effective. Sutcliffe et al., supra, at 661. For instance, 18of 30 peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes posttranslationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, I. A. et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

Non-limiting examples of antigenic polypeptides that can be used togenerate endokine-specific polyclonal and monoclonal antibodies includea polypeptide comprising or, alternatively, consisting of one, two,three or more of any of the following amino acid sequences andpolynucleotides encoding these polypeptides: amino acid residues fromabout 44 to about 158 in FIG. 1 (SEQ ID NO:2); amino acid residues fromabout 44 to about 54 in FIG. 1 (SEQ ID NO:2); amino acid residues fromabout 57 to about 68 in FIG. 1 (SEQ ID NO:2); amino acid residues fromabout 69 to about 78 in FIG. 1 (SEQ ID NO:2); amino acid residues fromabout 94 to about 105 in FIG. 1 (SEQ ID NO:2); amino acid residues fromabout 108 to about 132 in FIG. 1 (SEQ ID NO:2); and amino acid residuesfrom about 148 to about 158 in FIG. 1 (SEQ ID NO:2). As indicated above,the inventors have determined that the above polypeptide fragments areantigenic regions of the endokine alpha protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. See, Houghten, R. A., Proc. Natl.Acad. Sci. USA 82:5131-5135 (1985). This “Simultaneous Multiple PeptideSynthesis (SMPS)” process is further described in U.S. Pat. No.4,631,211 to Houghten et al. (1986). In this procedure the individualresins for the solid-phase synthesis of various peptides are containedin separate solvent-permeable packets, enabling the optimal use of themany identical repetitive steps involved in solid-phase methods. Acompletely manual procedure allows 500-1000 or more syntheses to beconducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemocyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde.

Animals such as rabbits, rats and mice are immunized with either free orcarrier-coupled peptides, for instance, by intraperitoneal and/orintradermal injection of emulsions containing about 100 μg peptide orcarrier protein and Freund's adjuvant. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalmay be increased by selection of anti-peptide antibodies, for instance,by adsorption to the peptide on a solid support and elution of theselected antibodies according to methods well known in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al (1984), supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art.

For instance, the immunologically important epitope in the coat proteinof foot-and-mouth disease virus was located by Geysen et al with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention which include, but are not limited to, Fab, Fab′ and F(ab′)2,Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes monoclonal, polyclonal, chimeric, humanized,and human monoclonal and polyclonal antibodies which specifically bindthe polypeptides of the present invention. The present invention furtherincludes antibodies which are anti-idiotypic to the antibodies of thepresent invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; and WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not to the method by whichit is produced. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlowet al., Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties).

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology and phage displaytechnology or through synthetic chemistry using methods known in theart. For example, the antibodies of the present invention can beprepared using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of a phage particle which carries polynucleotide sequencesencoding them. Phage with a desired binding property are selected from arepertoire or combinatorial antibody library (e.g. human or murine) byselecting directly with antigen, typically antigen bound or captured toa solid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman U. et al. (1995) J. Immunol. Methods182:41-50; Ames, R. S. et al. (1995) J. Immunol. Methods 184:177-186;Kettleborough, C. A. et al. (1994) Eur. J. Immunol. 24:952-958; Persic,L. et al. (1997) Gene 187 9-18; Burton, D. R. et al. (1994) Advances inImmunology 57:191-280; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO 95/20401; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727 and 5,733,743 (said references incorporated by reference intheir entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al.,BioTechniques 12(6):864-869 (1992); and Sawai, H. et al., AJRI 34:26-34(1995); and Better, M. et al., Science 240:1041-1043 (1988) (saidreferences incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88(1991); Shu, L.et al., PNAS 90:7995-7999 (1993); and Skerra, A. et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al., J.Immunol. Methods 125:191-202 (1989); and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0592 106; EP 0519596; Padlan E.A., Molecular Immunology 28(4/5):489-498 (1991); Studnicka G. M. et al.,Protein Engineering 7(6):805-814 (1994); Roguska M. A. et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patentapplication publication numbers WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said referencesincorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al., Immunol.Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies, S. O. et al.,PNAS 89:1428-1432 (1992); Fell, H. P. et al., J. Immunol. 146:2446-2452(1991) (said references incorporated by reference in their entireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal., PNAS 88:10535-10539 (1991); Zheng, X. X. et al., J. Immunol.154:5590-5600 (1994); and Vil, H. et al, PNAS 89:11337-11341 (1992)(said references incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng, B. et al., Blood 92(6):1981-1988(1998); Chen, Z. etal., CancerRes. 58(16):3668-3678 (1998); Harrop, J. A. et al., J.Immunol. 161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.160(7):3170-3179 (1998); Prat, M. et al., J. Cell Sci. 111(Pt2):237-247(1998); Pitard, V. et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard, J. et al., Cytokine 9(4):233-241 (1997); Carlson, N. G. etal., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman, R. E. et al.,Neuron 14(4):755-762 (1995); Muller, Y. A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al., Cytokine 8(1): 14-20 (1996)(saidreferences incorporated by reference in their entireties).

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

Epitopes

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in depositedclone [Deposit information] or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1 or containedin the clone deposited as ATCC Deposit Number 97640 on Jun. 27, 1996under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:1), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding, but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See,e.g., Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, e.g.,Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl.Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). A preferred immunogenic epitope includes thesecreted protein. The polypeptides comprising one or more immunogenicepitopes may be presented for eliciting an antibody response togetherwith a carrier protein, such as an albumin, to an animal system (suchas, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). If invivo immunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected by, for example, ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, and IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature 331:84-86 (1988). IgG fusion proteins thathave a disulfide-linked dimeric structure due to the IgG portiondesulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem. 270:3958-3964(1995). Nucleic acids encoding the above epitopes can also be recombinedwith a gene of interest as an epitope tag (e.g., the hemagglutinin(“HA”) tag or flag tag) to aid in detection and purification of theexpressed polypeptide. For example, a system described by Janknecht etal. allows for the ready purification of non-denatured fusion proteinsexpressed in human cell lines (Janknecht et al., Proc. Natl. Acad. Sci.USA 88:8972-897 (1991)). In this system, the gene of interest issubcloned into a vaccinia recombination plasmid such that the openreading frame of the gene is translationally fused to an amino-terminaltag consisting of six histidine residues. The tag serves as amatrix-binding domain for the fusion protein. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; and WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; and 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M,10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M,5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M,5×10⁻¹⁵M, and 10⁻¹⁵M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding, butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibodyagonists can be made using methods known in the art. See, e.g., PCTpublication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood92(6): 1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell Sci. 111(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301(1997); Taryman et al., Neuron 14(4):755-762(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory Press, 2nd ed. 1988) (incorporated by reference herein in itsentirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; and WO 89/12624; U.S. Pat. No. 5,314,995; andEP 396,387.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include, butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties). The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 3. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958(1994); Persic et al., Gene 187:9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques4:214(1986); Gillies et al., J. Immunol. Methods 125:191-202(1989); U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (Int. Rev. Immunol. 13:65-93 (1995)). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated therefrom, or nucleic acid,preferably poly A+ RNA, isolated therefrom, or any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (See, for example, the techniques described inSambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel etal., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1998), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54(1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5): 155-215 (May 1993)); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al., eds, Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150: 1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, “The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells,” in DNA Cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol Cell Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA77:2197 (1980)). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432(1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988)). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitates theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. eds., pp. 243-56 (Alan R. Liss, Inc.1985); Hellstrom et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (MarcelDekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. eds., pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. eds., pp. 303-16 (AcademicPress 1985), and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58(1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include, but are not limited to, competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York (1994), which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel(et al., eds, Current Protocols in Molecular Biology, Vol. 1, John Wiley& Sons, Inc., New York (19914) at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubel,et al., eds, Current Protocols in Molecular Biology, Vol. 1, John Wiley& Sons, Inc., New York (1994) at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel, et al., eds, Current Protocols in Molecular Biology, Vol. 1,John Wiley & Sons, Inc., New York (1994) at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 1251) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., 3H or1251) in the presence of increasing amounts of an unlabeled secondantibody.

Endokine Alpha Related Disorder Diagnosis

Endokine alpha is a new member of the TNF family of cytokines. Forendokine alpha related disorders, it is believed that substantiallyaltered (increased or decreased) levels of endokine alpha geneexpression can be detected in tissue or other cells or bodily fluids(e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” endokinealpha gene expression level, that is, the endokine alpha expressionlevel in tissue or bodily fluids from an individual not having thedisorder. Thus, the invention provides a diagnostic method useful duringdiagnosis of an endokine alpha-related disorder, which involvesmeasuring the expression level of the gene encoding the endokine alphaprotein in tissue or other cells or body fluid from an individual andcomparing the measured gene expression level with a standard endokinealpha gene expression level, whereby an increase or decrease in the geneexpression level compared to the standard is indicative of an endokinealpha related disorder.

By individual is intended mammalian individuals, preferably humans. By“measuring the expression level of the gene encoding the endokine alphaprotein” is intended qualitatively or quantitatively measuring orestimating the level of the endokine alpha protein or the level of themRNA encoding the endokine alpha protein in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to the endokinealpha protein level or mRNA level in a second biological sample).Preferably, the endokine alpha protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardendokine alpha protein level or mRNA level, the standard being takenfrom a second biological sample obtained from an individual not havingthe disorder or being determined by averaging levels from a populationof individuals not having a disorder involving endokine alpha. As willbe appreciated in the art, once a standard endokine alpha protein levelor mRNA level is known, it can be used repeatedly as a standard forcomparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains endokine alpha protein or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovial fluidand spinal fluid) which contain secreted mature endokine alpha protein,or tissue sources found to express endokine alpha. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

The present invention is useful for diagnosis of various endokinealpha-related disorders in mammals, preferably humans, as similar toTNF-like disorders known in the art or as presented herein. Theseinclude disorders associated with immunomodulation and inflammation,cell proliferation, angiogenesis, tumor metastases, apoptosis, sepsisand endotoxemia.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as thesingle-step-guanidinium-thiocyanate-phenol-chloro form method describedin Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding an endokine alpha polypeptide are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.,Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxaudimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. Endokine alpha proteincDNA labeled according to any appropriate method (such as the³²P-multiprimed DNA labeling system (Amersham)) is used as probe. Afterhybridization overnight, the filter is washed and exposed to x-ray film.cDNA for use as probe according to the present invention is described inthe sections above and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe endokine alpha protein). Northern blot analysis can be performed asdescribed above.

Preferably, levels of mRNA encoding the endokine alpha protein areassayed using the RT-PCR method described in Makino et al., Technique2:295-301 (1990). By this method, the radioactivities of the “amplicons”in the polyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the endokine alphaprotein) is quantified using an imaging analyzer. RT and PCR reactioningredients and conditions, reagent and gel concentrations, and labelingmethods are well known in the art. Variations on the RT-PCR method willbe apparent to the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying endokine alpha protein levels in a biological sample can occurusing any art-known method. Preferred for assaying endokine alphaprotein levels in a biological sample are antibody-based techniques. Forexample, endokine alpha protein expression in tissues can be studiedwith classical immunohistological methods. In these, the specificrecognition is provided by the primary antibody (polyclonal ormonoclonal), but the secondary detection system can utilize fluorescent,enzyme, or other conjugated secondary antibodies. As a result, animmunohistological staining of tissue section for pathologicalexamination is obtained. Tissues can also be extracted, e.g., with ureaand neutral detergent, for the liberation of endokine alpha protein forWestern-blot or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987)). In this technique, which is based on the use of cationic solidphases, quantitation of endokine alpha protein can be accomplished usingisolated endokine alpha protein as a standard. This technique can alsobe applied to body fluids. With these samples, a molar concentration ofendokine alpha protein will aid to set standard values of endokine alphaprotein content for different body fluids, like serum, plasma, urine,synovial fluid, spinal fluid, etc. The normal appearance of endokinealpha protein amounts can then be set using values from healthyindividuals, which can be compared to those obtained from a testsubject.

Other antibody-based methods useful for detecting endokine alpha proteinlevels include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). For example, endokinealpha protein-specific monoclonal antibodies can be used both as animmunoadsorbent and as an enzyme-labeled probe to detect and quantifythe endokine alpha protein. The amount of endokine alpha protein presentin the sample can be calculated by reference to the amount present in astandard preparation using a linear regression computer algorithm. Suchan ELISA for detecting a tumor antigen is described in Iacobelli et al.,Breast Cancer Research and Treatment 11:19-30 (1988). In another ELISAassay, two distinct specific monoclonal antibodies can be used to detectendokine alpha protein in a body fluid. In this assay, one of theantibodies is used as the immunoadsorbent and the other as theenzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting endokinealpha protein with immobilized antibody and, without washing, contactingthe mixture with the labeled antibody. The “two-step” assay involveswashing before contacting the mixture with the labeled antibody. Otherconventional methods may also be employed as suitable. It is usuallydesirable to immobilize one component of the assay system on a support,thereby allowing other components of the system to be brought intocontact with the component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

In addition to assaying endokine alpha protein levels in a biologicalsample obtained from an individual, endokine alpha protein can also bedetected in vivo by imaging. Antibody labels or markers for in vivoimaging of endokine alpha protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A endokine alpha protein-specific antibody or antibody portion which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹¹In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorder. Itwill be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moietiesneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of ^(99m)Tc. The labeledantibody or antibody portion will then preferentially accumulate at thelocation of cells which contain endokine alpha protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Portions” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, Burchiel, S. W. andRhodes, B. A. eds., Masson Publishing Inc. (1982)).

Endokine alpha-protein specific antibodies for use in the presentinvention can be raised against the intact endokine alpha protein or anantigenic polypeptide portion thereof, which may presented together witha carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody portions (suchas, for example, Fab and F(ab′)₂ portions) which are capable ofspecifically binding to endokine alpha protein. Fab and F(ab′)₂ portionslack the Fc portion of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, theseportions are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the endokine alphaprotein or an antigenic portion thereof can be administered to an animalin order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of endokine alphaprotein is prepared and purified as described above to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or endokine alpha protein binding portionsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (see, e.g., Colligan, Current Protocols in Immunology, WileyInterscience, New York (1990-1996); Harlow & Lane, Antibodies: ALaboratory Manual, Chs. 6-9, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1988); Ausubel, infra, at Chapter 11, these referencesentirely incorporated herein by reference).

In general, such procedures involve immunizing an animal (preferably amouse) with an endokine alpha polypeptide antigen or with an endokinealpha polypeptide-expressing cell. Suitable cells can be recognized bytheir capacity to bind anti-endokine alpha protein antibody. Such cellsmay be cultured in any suitable tissue culture medium (e.g., Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), supplemented with about 10 μg/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin). The splenocytes of such mice are extracted andfused with a suitable myeloma cell line. Any suitable myeloma cell linemay be employed in accordance with the present invention (e.g., parentmyeloma cell line (SP₂O), available from the American Type CultureCollection (ATCC) (Manassas, Va., USA)). After fusion, the resultinghybridoma cells are selectively maintained in HAT medium, and thencloned by limiting dilution as described by Wands et al.,Gastroenterology 80:225-232 (1981); Harlow & Lane, infra, Chapter 7. Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the endokinealpha antigen.

Alternatively, additional antibodies capable of binding to the endokinealpha protein antigen may be produced in a two-step procedure throughthe use of anti-idiotypic antibodies. Such a method makes use of thefact that antibodies are themselves antigens, and therefore it ispossible to obtain an antibody which binds to a second antibody. Inaccordance with this method, endokine alpha protein specific antibodiesare used to immunize an animal, preferably a mouse. The splenocytes ofsuch an animal are then used to produce hybridoma cells, and thehybridoma cells are screened to identify clones which produce anantibody whose ability to bind to the endokine alpha protein-specificantibody can be blocked by the endokine alpha protein antigen. Suchantibodies comprise anti-idiotypic antibodies to the endokine alphaprotein-specific antibody and can be used to immunize an animal toinduce formation of further endokine alpha protein-specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other portions of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such portions are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab portions) orpepsin (to produce F(ab′)₂ portions). Alternatively, endokine alphaprotein-binding portions can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Where in vivo imaging is used to detect enhanced levels of endokinealpha protein for diagnosis in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

Further suitable labels for the endokine alpha protein-specificantibodies of the present invention are provided below. Examples ofsuitable enzyme labels include malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In and ^(99m)Tc are preferredisotopes where in vivo imaging is used since they avoid the problem ofdehalogenation of the ¹²⁵I or ¹³¹I-labeled monoclonal antibody by theliver. In addition, these radionucleotides have a more favorable gammaemission energy for imaging (Perkins et al., Eur. J. Nucl. Med.10:296-301 (1985); Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)).For example, ¹¹¹In coupled to monoclonal antibodies with1-(p-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumoroustissues, particularly the liver, and therefore enhances specificity oftumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and Fe.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al (Clin. Chim. Acta 70:1-31 (1976)), andSchurs et al. (Clin. Chim. Acta 81:1-40 (1977)). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a VL or VH domain. The antibodiesmay be from any animal origin including birds and mammals. Preferably,the antibodies are human, murine, rabbit, goat, guinea pig, camel,horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodieswhich are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893,5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al., J. Immunol.148:1547-1553 (1992). Antibodies of the present invention may bedescribed or specified in terms of the epitope(s) or portion(s) of apolypeptide of the present invention which are recognized orspecifically bound by the antibody. The epitope(s) or polypeptideportion(s) may be specified as described herein, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues, orlisted in the Tables and Figures. Antibodies which specifically bind anyepitope or polypeptide of the present invention may also be excluded.Therefore, the present invention includes antibodies that specificallybind polypeptides of the present invention, and allows for the exclusionof the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. Monoclonal antibodies can be prepared using a wide oftechniques known in the art including the use of hybridoma andrecombinant technology. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: Monoclonal Antibodies and T-cell Hybridomas, pp.563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties).

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al., J. Immunol. Methods 182:41-50 (1995); Ames, R. S. et al., J.Immunol. Methods 184:177-186 (1995); Kettleborough, C. A. et al., Eur.J. Immunol. 24:952-958 (1994); Persic, L. et al., Gene 187:9-18 (1997);Burton, D. R. et al., Advances in Immunology 57:191-280(1994);PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al.,BioTechniques 12(6):864-869 (1992); and Sawai, H. et al., AJRI 34:26-34(1995); and Better, M. et al., Science 240:1041-1043 (1988) (saidreferences incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al., PNAS 90:7995-7999 (1993); and Skerra, A. et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al., J.Immunol. Methods 125:191-202 (1989); and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., Molecular Immunology 28(4/5):489-498 (1991); Studnicka G. M. etal., Protein Engineering 7(6):805-814 (1994); Roguska M. A. et al., PNAS91:969-973) (1994), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645(said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al., Immunol.Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies, S. O. et al.,PNAS 89:1428-1432 (1992); Fell, H. P. et al., J. Immunol. 146:2446-2452(1991) (said references incorporated by reference in their entireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal., PNAS 88:10535-10539 (1991); Zheng, X. X. et al., J. Immunol.154:5590-5600 (1995); and Vil, H. et al., PNAS 89:11337-11341 (1992)(said references incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding, butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. see e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng, B. et al., Blood 92(6):1981-1988(1998); Chen, Z. etal., Cancer Res. 58(16):3668-3678 (1998); Harrop, J. A. et al., J.Immunol. 161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci. 111(Pt2):237-247(1998); Pitard, V. et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard, J. et al., Cytokinde 9(4):233-241 (1997); Carlson, N. G. etal., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman, R. E. et al.Neuron 14(4):755-762 (1995); Muller, Y. A. et al., Structure6(9):1153-1167 (1998); Bartunek, P. et al., Cytokine 8(1): 14-20 (1996)(said references incorporated by reference in their entireties).

Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, Intl. Rev. Cytol.115:171-229 (1989), which is incorporated by reference herein in itsentirety. Further, the contents of each of the documents recited in thisparagraph is herein incorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the polynucleotide transgene be integratedinto the chromosomal site of the endogenous gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al, Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of endokine alpha polypeptides,studying conditions and/or disorders associated with aberrant endokinealpha expression, and in screening for compounds effective inameliorating such conditions and/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Thecells are genetically engineered in vitro using recombinant DNAtechniques to introduce the coding sequence of polypeptides of theinvention into the cells, or alternatively, to disrupt the codingsequence and/or endogenous regulatory sequence associated with thepolypeptides of the invention, e.g., by transduction (using viralvectors, and preferably vectors that integrate the transgene into thecell genome) or transfection procedures, including, but not limited to,the use of plasmids, cosmids, YACs, naked DNA, electroporation,liposomes, etc. The coding sequence of the polypeptides of the inventioncan be placed under the control of a strong constitutive or induciblepromoter or promoter/enhancer to achieve expression, and preferablysecretion, of the polypeptides of the invention. The engineered cellswhich express and preferably secrete the polypeptides of the inventioncan be introduced into the patient systemically, e.g., in thecirculation, or intraperitoneally. Alternatively, the cells can beincorporated into a matrix and implanted in the body, e.g., geneticallyengineered fibroblasts can be implanted as part of a skin graft;genetically engineered endothelial cells can be implanted as part of alymphatic or vascular graft. (See, for example, Anderson et al. U.S.Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, eachof which is incorporated by reference herein in its entirety. See alsoU.S. Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative SelectionMethods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi, et al., Cellsand Non-Human Organisms Containing Predetermined Genomic Modificationsand Positive-Negative Selection Methods and Vectors for Making Same);U.S. Pat. No. 4,736,866 (Leder, et al., Transgenic Non-Human Animals);and U.S. Pat. No. 4,873,191 (Wagner, et al., Genetic Transformation ofZygotes); each of which is hereby incorporated by reference in itsentirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Antagonists

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 97640. In one embodiment,antisense sequence is generated internally by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the endokine alpha antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector or a portion thereof, is transcribed,producing an antisense nucleic acid (RNA) of the invention. Such avector would contain a sequence encoding the endokine alpha antisensenucleic acid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others know in the art, used for replication and expression invertebrate cells. Expression of the sequence encoding endokine alpha, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, Nature 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinster,et al., Nature 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of an endokinealpha gene. However, absolute complementarity, although preferred, isnot required. A sequence “complementary to at least a portion of anRNA,” referred to herein, means a sequence having sufficientcomplementarity to be able to hybridize with the RNA, forming a stableduplex; in the case of double stranded endokine alpha antisense nucleicacids, a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid Generally, the larger the hybridizing nucleic acid, the more basemismatches with an endokine alpha RNA it may contain and still form astable duplex (or triplex as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of the nucleotide sequence shownin FIG. 1 could be used in an antisense approach to inhibit translationof endogenous endokine alpha mRNA. Oligonucleotides complementary to the5′ untranslated region of the mRNA should include the complement of theAUG start codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of endokine alpha mRNA, antisense nucleicacids should be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (see,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (see, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, a-D-galactosylqueosine,inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al., Nucl. Acids Res. 16:3209 (1988),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al, Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the endokine alpha codingregion sequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (see, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy endokine alpha mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of endokine alpha (FIG. 1). Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the endokine alpha mRNA; i.e., to increase efficiencyand minimize the intracellular accumulation of non-functional mRNAtranscripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express endokinealpha in vivo. DNA constructs encoding the ribozyme may be introducedinto the cell in the same manner as described above for the introductionof antisense encoding DNA. A preferred method of delivery involves usinga DNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous endokine alpha messages and inhibittranslation. Since ribozymes unlike antisense molecules, are catalytic,a lower intracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the endokine alpha gene and/or its promoter usingtargeted homologous recombination. (e.g., see Smithies et al., Nature317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompsonet al., Cell 5:313-321 (1989); each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functionalpolynucleotide of the invention (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous polynucleotide sequence(either the coding regions or regulatory regions of the gene) can beused, with or without a selectable marker and/or a negative selectablemarker, to transfect cells that express polypeptides of the invention invivo. In another embodiment, techniques known in the art are used togenerate knockouts in cells that contain, but do not express the gene ofinterest. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the targeted gene. Suchapproaches are particularly suited in research and agricultural fieldswhere modifications to embryonic stem cells can be used to generateanimal offspring with an inactive targeted gene (e.g., see Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art. The contents of each of the documents recited in thisparagraph is herein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present inventioninclude soluble forms of endokine alpha (e.g., fragments of the endokinealpha polypeptide shown in FIG. 1 that include the ligand binding domainfrom the extracellular region of the full length receptor). Such solubleforms of endokine alpha, which may be naturally occurring or synthetic,antagonize endokine alpha mediated signaling by competing with the cellsurface bound forms of the receptor for binding to TNF-family ligands.Antagonists of the present invention also include antibodies specificfor TNF-family ligands and endokine alpha-Fc fusion proteins.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing and/or blocking the ligand/receptorsignaling pathway. Members of the TNF ligand family include, but are notlimited to, TNF-a, lymphotoxin-a (LT-a, also known as TNF-b), LT-b(found in complex heterotrimer LT-a2-b), FasL, CD40L, CD27L, CD30L,4-1BBL, OX40L and nerve growth factor (NGF).

TNF-α has been shown to protect mice from infection with herpes simplexvirus type 1 (HSV-1). Rossol-Voth et al., J. Gen. Virol. 72:143-147(1991). The mechanism of the protective effect of TNF-α is unknown butappears to involve neither interferons nor NK cell killing. One memberof the TNFR family has been shown to mediate HSV-1 entry into cells.Montgomery et al., Eur. Cytokine Newt. 7:159 (1996). Further, antibodiesspecific for the extracellular domain of this TNFR block HSV-1 entryinto cells. Thus, endokine alpha antagonists of the present inventioninclude both endokine alpha amino acid sequences and antibodies capableof preventing TNFR mediated viral entry into cells. Such sequences andantibodies can function by either competing with cell surface localizedTNFR for binding to virus or by directly blocking binding of virus tocell surface receptors.

Antibodies according to the present invention may be prepared by any ofa variety of standard methods using endokine alpha receptor immunogensof the present invention. Such endokine alpha receptor immunogensinclude the endokine alpha receptor protein shown in FIG. 1 (SEQ IDNO:2) (which may or may not include a leader sequence) and polypeptidefragments of the receptor comprising the ligand binding, extracellular,transmembrane, the intracellular domains of the endokine alphareceptors, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedherein and/or known in the art, such as, for example, those methodsdescribed in Tartaglia and Goeddel, J. Biol. Chem.267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216 (1993)), andPCT Application WO 94/09137 (the contents of each of these threeapplications are herein incorporated by reference in their entireties),and are preferably specific to polypeptides of the invention having theamino acid sequence of SEQ ID NO:2.

As one of skill in the art will appreciate, endokine alpha polypeptidesof the present invention and the epitope-bearing fragments thereofdescribed above can be combined with heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, and IgM) orportions thereof (CH1, CH2, CH3, and any combination thereof, includingboth entire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life.

The techniques of gene-shuffling, motif-shuffling, exon-shuffling,and/or codon-shuffling (collectively referred to as “DNA shuffling”) maybe employed to modulate the activities of endokine alpha therebyeffectively generating agonists and antagonists of endokine alpha. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of endokine alpha polynucleotides andcorresponding polypeptides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments into adesired endokine alpha molecule by homologous, or site-specific,recombination. In another embodiment, endokine alpha polynucleotides andcorresponding polypeptides may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. In another embodiment, one or morecomponents, motifs, sections, parts, domains, fragments, etc., ofendokine alpha may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules are, forexample, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J.Exp. Med. 188(6):1185-1190), endokine-alpha (International PublicationNos. WO 98/07880 and WO 98/18921), OPG, OX40, nerve growth factor (NGF),and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), TR12, and TNF-R1,TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1/DR4/APO-2, TRAIL-R2/DR5,DcR1/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, and v-FLIP.

In further preferred embodiments, the heterologous molecules are anymember of the TNF family.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an endokine alpha proteingene. This can be accomplished using a variety of well known techniquesand libraries, which generally are available commercially. The genomicDNA then is used for in situ chromosome mapping using well knowntechniques for this purpose. Typically, in accordance with routineprocedures for chromosome mapping, some trial and error may be necessaryto identify a genomic probe that gives a good in situ hybridizationsignal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified portion.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of portions from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Endokine Alpha Uses

The Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes(Goeddel, D. V. et al., “Tumor Necrosis Factors: Gene Structure andBiological Activities,” Symp. Quant. Biol. 51:597-609 (1986), ColdSpring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem.57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988); Fiers, W.,FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce suchvarious cellular responses by binding to TNF-family receptors.

Endokine alpha polynucleotides, polypeptides, agonists or antagonists ofthe invention may be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof endokine alpha. Endokine alpha polypeptides, agonists or antagonistsmay be administered to a patient (e.g., mammal, preferably human)afflicted with such a disorder. Alternatively, a gene therapy approachmay be applied to treat such disorders. Disclosure herein of endokinealpha nucleotide sequences permits the detection of defective endokinealpha genes, and the replacement thereof with normal endokinealpha-encoding genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparision of the endokine alpha nucleotidesequence disclosed herein with that of a endokine alpha gene derivedfrom a patient suspected of harboring a defect in this gene.

In another embodiment, the polypeptides of the present invention areused as a research tool for studying the biological effects that resultfrom inhibiting TR11/endokine alpha interactions on different celltypes. endokine alpha polypeptides also may be employed in in vitroassays for detecting TR11 or endokine alpha or the interactions thereof.

As indicated above, TNF is noted for its pro-inflammatory actions whichresult in tissue injury, such as induction of procoagulant activity onvascular endothelial cells (Pober, J. S. et al., J. Immunol. 136:1680(1986)), increased adherence of neutrophils and lymphocytes (Pober, J.S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the releaseof platelet activating factor from macrophages, neutrophils and vascularendothelial cells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).Recent evidence implicates TNF in the pathogenesis of many infections(Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders,neoplastic pathology, e.g., in cachexia accompanying some malignancies(Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologiesand graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med.166:1280 (1987)). A number of studies have suggested that TNF is animportant mediator of the cachexia in cancer, infectious pathology, andin other catabolic states.

Thus, the endokine alpha protein of the present invention can be usedfor tumor targeting, preferably, after conjugation with radioisotypes orcytostatic drugs (Gruss and Dower, Blood 85(12):3378-3404 (1995)).Endokine alpha can be used in patients with melanoma and sarcoma fortumor regression and extension of patient life span through a localinjection or used in isolated limb perfusion (Aggarwal and Natarajan,Eur. Cytokine Netw. 7(2):92-124 (1996)).

The endokine alpha of the present invention can also have a therapeuticrole in specific situations, for example, activity against viral,bacterial, yeast, fungal, and other infections (including toxoplasmagondii, schistosoma mansoni, listeria monocytogens and BCG). Theseeffects of endokine alpha can be indirect and thus preferably, mediatedthrough activation of macrophages, eosinophils, fibroblasts, orneutrophils.

TNF is also thought to play a central role in the pathophysiologicalconsequences of Gram-negative sepsis and endotoxic shock (Michie, H. R.et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., SecondVienna Shock Forum, p. 463-466 (1989); Simpson, S. Q. et al., Crit. CareClin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.Endotoxin is a potent monocyte/macrophage activator which stimulatesproduction and secretion of TNF (Kombluth, S. K. et al., J. Immunol.137:2585-2591 (1986)) and other cytokines. Elevated levels ofcirculating TNF have also been found in patients suffering fromGram-negative sepsis (Waage, A. et al., Lancet 1:355-357 (1987);Hammerle, A. F. et al., Second Vienna Shock Forum p. 715-718(1989);Debets, J. M. H. et al., Crit. Care Med. 17:489-497 (1989); Calandra, T.et al., J. Infec. Dis. 161:982-987 (1990)).

Neutralizing antisera or mAbs to TNF have been shown in mammals otherthan man to abrogate adverse phaysiological changes and prevent deathafter lethal challenge in experimental endotoxemia and bacteremia. Thiseffect has been demonstrated, e.g., in rodent lethality assays and inprimate pathology model systems (Mathison, J. C. et al., J. Clin.Invest. 81:1925-1937 (1988); Beutler, B. et al., Science 229:869-871(1985); Tracey, K. J. et al., Nature 330:662-664 (1987); Shimamoto, Y.et al., Immunol. Lett. 17:311-318 (1988); Silva, A. T. et al., J.Infect. Dis. 162:421-427 (1990); Opal, S. M. et al., J. Infect. Dis.161:1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279-292(1990)). To date, experience with anti-TNF mAb therapy in humans hasbeen limited but shows beneficial therapeutic results, e.g., inarthritis and sepsis. See, e.g., Elliott, M. J. et al., Baillieres Clin.Rheumatol. 9:633-52 (1995); Feldmann M, et al., Ann. N.Y. Acad. Sci. USA766:272-8 (1995); van der Poll, T. et al., Shock 3:1-12 (1995); Wherryet al., Crit. Care. Med. 21:S436-40 (1993); Tracey K. J., et al., Crit.Care Med. 21:S415-22 (1993).

As endokine alpha is believed to exhibit many of the biological effectsof TNF, the present invention is further directed to antibody-basedtherapies which involve administering an anti-endokine alpha antibody toa mammalian, preferably human, patient for treating one or more of theabove-described disorders. Methods for producing anti-endokine alphapolyclonal and monoclonal antibodies are described in detail above. Suchantibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein.

Polynucleotides and/or polypeptides of the invention, and/or agonistsand/or antagonists thereof, are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e.g., immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, gliobalstoma, cancer associated with mutation oralteration of p53, brain tumor, bladder cancer, uterocervical cancer,colon cancer, colorectal cancer, non-small cell carcinoma of the lung,small cell carcinoma of the lung, stomach cancer, etc.),lymphoproliferative disorders (e.g., lymphadenopathy and lymphomas(e.g., EBV induced lymphoproliferations and Hodgkin's disease),microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1infection, HIV-2 infection, herpesvirus infection (including, but notlimited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirusinfection, poxvirus infection, human papilloma virus infection,hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pyloriinfection, invasive Staphylococcia, etc.), parasitic infection,nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain,cardiovascular disorders (e.g., neovascularization, hypovascularizationor reduced circulation (e.g., ischemic disease (e.g., myocardialinfarction, stroke, etc.)), AIDS, allergy, inflammation,neurodegenerative disease (e.g., Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellardegeneration, etc.), graft rejection (acute and chronic), graft vs. hostdisease, diseases due to osteomyelodysplasia (e.g., aplastic anemia,etc.), joint tissue destruction in rheumatism, liver disease (e.g.,acute and chronic hepatitis, liver injury, and cirrhosis), autoimmunedisease (e.g., multiple sclerosis, myasthenia gravis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, inflammatoryautoimmune diseases, etc.), cardiomyopathy (e.g., dilatedcardiomyopathy), diabetes, diabetic complications (e.g., diabeticnephropathy, diabetic neuropathy, diabetic retinopathy), influenza,asthma, psoriasis, glomerulonephritis, septic shock, and ulcerativecolitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures), and regulating boneformation and treating osteoporosis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are also useful as an adjuvant to enhanceimmune responsiveness to specific antigen and/or anti-viral immuneresponses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders or in the prevention of transplant rejection. Inspecific embodiments, polynucleotides and/or polypeptides of theinvention are used to treat or prevent chronic inflammatory, allergic orautoimmune conditions, such as those described herein or are otherwiseknown in the art.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding endokine alpha locally orsystemically in the body or by direct cytotoxicity of the antibody,e.g., as mediated by complement (CDC) or by effector cells (ADCC). Someof these approaches are described in more detail below. Armed with theteachings provided herein, one of ordinary skill in the art will knowhow to use the antibodies of the present invention for diagnostic,monitoring or therapeutic purposes without undue experimentation.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Amountsand regimens for the administration of antibodies, their fragments orderivatives can be determined readily by those with ordinary skill inthe clinical art of treating TNF-related disease.

For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, or buccalroutes. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein the antibody, fragment or derivative is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. The effective dose is a function of theindividual chimeric or monoclonal antibody, the presence and nature of aconjugated therapeutic agent (see below), the patient and his clinicalstatus, and can vary from about 10 μg/kg body weight to about 5000 mg/kgbody weight. The preferred dosages comprise 0.1 to 500 mg/kg body wt.

In addition to the pharmacologically active compounds, the newpharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations contain fromabout 0.01 to 99 percent, preferably from about 20 to 75 percent ofactive compound(s), together with the excipient.

Similarly, preparations of an endokine alpha antibody or fragment of thepresent invention for parenteral administration, such as in detectablylabeled form for imaging or in a free or conjugated form for therapy,include sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present, such as, forexample, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. See, generally, Remington's Pharmaceutical Science,16th ed., Mack Publishing Co., Easton, Pa., 1980.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developingendokine alpha related disorders as described herein. Such treatmentcomprises parenterally administering single or multiple doses of theantibody, a fragment or derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hemopoietic growth factors, etc., which serve to increasethe number or activity of effector cells which interact with theantibodies.

Since circulating concentrations of endokine alpha (like TNF) tend to beextremely low, in the range of about 10 pg/ml in non-septic individuals,and reaching about 50 pg/ml in septic patients and above 100 pg/ml inthe sepsis syndrome for TNF (Hammerle, A. F. et al., 1989, supra) or maybe only be detectable at sites of endokine alpha-related disorders, itis preferred to use high affinity and/or potent in vivo endokinealpha-inhibiting and/or neutralizing antibodies, fragments or regionsthereof, for both endokine alpha immunoassays and therapy of endokinerelated disorders. Such antibodies, fragments, or regions, willpreferably have an affinity for human endokine alpha, expressed as Ka,of at least 10⁸ M⁻¹, more preferably, at least 10⁹ M⁻¹, such as 5×10⁸M⁻¹, 8×10⁸ M⁻¹, 2×10⁹ M⁻¹, 4×10⁹ M⁻¹, 6×10⁹ M⁻¹, 8×10⁹ M⁻¹.

Preferred for human therapeutic use are high affinity murine andmurine/human or human/human chimeric antibodies, and fragments, regionsand derivatives having potent in vivo endokine-inhibiting and/orneutralizing activity, according to the present invention, e.g., thatblock endokine-induced IL-1, IL-6 or TNF secretion, procoagulantactivity, expression of cell adhesion molecules such as ELAM-1 andICAM-1 and mitogenic activity, in vivo, in situ, and in vitro.

Additional preferred embodiments of the invention include, but are notlimited to, the use of endokine-α polypeptides and functional agonistsin the following applications:

A vaccine adjuvant that enhances immune responsiveness to specificantigen.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses.

As a stimulator of B cell responsiveness to pathogens.

As a activator of T cells.

As an agent that elevates the immune status of a individual prior totheir receipt of immunosuppressive therapies.

As an agent to accelerate recovery of immunocompromised individuals; Asan agent to boost immunoresponsiveness among aged populations; As animmune system enhancer following bone marrow transplant.

As an agent to direct an individuals immune system towards developmentof a humoral response (i.e. TH2) and/or a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example multiple myeloma is aslowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

As a B cell and other ligand expressing cell (e.g., endothelial cells)specific binding protein to which specific activators or inhibitors ofcell growth may be attached. The result would be to focus the activityof such activators or inhibitors onto normal, diseased, or neoplastic Bcell populations.

As a means of detecting B-lineage cells and/or ligand expressing cells(e.g., endothelial cells) by virtue of its specificity. This applicationmay require labeling the protein with biotin or other agents to afford ameans of detection.

As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodificiency;

As part of a B cell selection device the function of which is to isolateB cells as well as other ligand expressing cells (e.g., endothelialcells) from a heterogenous mixture of cell types. Endokine alpha couldbe coupled to a solid support to which B cells would then specificallybind. Unbound cells would be washed out and the bound cells subsequentlyeluted. This technique would allow purging of tumor cells from, forexample, bone marrow or peripheral blood prior to transplant.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceendokine alpha mediated responses.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecovery.

As a means of regulating secreted cytokines that are elicited byendokine alpha.

Inhibition of unwanted TH1 responses. There is strong evidence todemonstrate that IL-12 is an important cytokine in directing TH1differentiation. Endokine alpha-induced inhibition of IL-12 productionmight be helpful in controlling TH1-associated conditions, such asautoimmune diseases, inflammation, acute allograft rejection, fetalreabsorption.

Reduction of inflammatory response. It was shown that induction of IL-10and MCP-1 ameliorate experimental fecal peritonitis.

Enhanced resistance to pathogens. Products of the oxidative burst, suchas H₂O₂, are used by monocytes for the killing of phagocytosed pathogensor for the extracellular destruction of cells.

All of the above described applications as they may apply to veterinarymedicine.

Antagonists of endokine alpha include binding and/or inhibitoryantibodies, antisense nucleic acids, ribozymes or soluble forms of theendokine alpha receptor(s). These would be expected to reverse many ofthe activities of the ligand described above as well as find clinical orpractical application as:

A means of blocking various aspects of immune responses to foreignagents or self. Examples include autoimmune disorders such as lupus, andarthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens. Although our currentdata speaks directly to the potential role of endokine alpha in B cell,T cell and monocyte related pathologies, it remains possible that othercell types may gain expression or responsiveness to endokine alpha.Thus, endokine alpha may, like CD40 and its ligand, be regulated by thestatus of the immune system and the microenvironment in which the cellis located.

A therapy for preventing the B and/or T cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythramatosus and MS.

An inhibitor of B and or T cell migration in endothelial cells, Thisactivity disrupts tissue architecture or cognate responses and isuseful, for example, in disrupting immune responses, and blockingsepsis.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell and/or T cell malignancies such as ALL, Hodgkinsdisease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia,plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformeddiseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseasesas monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, and related idiopathic monoclonalgammopathies.

A means of decreasing the involvement of B cells and Ig associated withChronic Myelogenous Leukemia.

An immunosuppressive agent(s).

An inhibitor of signaling pathways involving ERK1, COX2 and Cyclin D2which have been associated with endokine alpha induced B cellactivation.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit endokine alphachemotaxis and activation of macrophages and their precursors, and ofneutrophils, basophils, B lymphocytes and some T-cell subsets, e.g.,activated and CD8 cytotoxic T cells and natural killer cells, in certainauto-immune and chronic inflammatory and infective diseases. Examples ofauto-immune diseases include multiple sclerosis, and insulin-dependentdiabetes.

The antagonists may also be employed to treat infectious diseasesincluding silicosis, sarcoidosis, idiopathic pulmonary fibrosis bypreventing the recruitment and activation of mononuclear phagocytes.They may also be employed to treat idiopathic hyper-eosinophilicsyndrome by preventing eosinophil production and migration. Endotoxicshock may also be treated by the antagonists by preventing the migrationof macrophages and their production of the endokine alpha polypeptidesof the present invention. The antagonists may also be employed fortreating atherosclerosis, by preventing monocyte infiltration in theartery wall.

The antagonists may also be employed to treat histamine-mediatedallergic reactions and immunological disorders including late phaseallergic reactions, chronic urticaria, and atopic dermatitis byinhibiting chemokine-induced mast cell and basophil degranulation andrelease of histamine. IgE-mediated allergic reactions such as allergicasthma, rhinitis, and eczema may also be treated.

The antagonists may also be employed to treat chronic and acuteinflammation by preventing the attraction of monocytes to a wound area.They may also be employed to regulate normal pulmonary macrophagepopulations, since chronic and acute inflammatory pulmonary diseases areassociated with sequestration of mononuclear phagocytes in the lung.Antagonists may also be employed to treat rheumatoid arthritis bypreventing the attraction of monocytes into synovial fluid in the jointsof patients. Monocyte influx and activation plays a significant role inthe pathogenesis of both degenerative and inflammatory arthropathies.The antagonists may be employed to interfere with the deleteriouscascades attributed primarily to IL-1 and TNF, which prevents thebiosynthesis of other inflammatory cytokines. In this way, theantagonists may be employed to prevent inflammation. The antagonists mayalso be employed to inhibit prostaglandin-independent fever induced byendokine alpha. The antagonists may also be employed to treat cases ofbone marrow failure, for example, aplastic anemia and myelodysplasticsyndrome. The antagonists may also be employed to treat asthma andallergy by preventing eosinophil accumulation in the lung. Theantagonists may also be employed to treat subepithelial basementmembrane fibrosis which is a prominent feature of the asthmatic lung.

Antibodies against endokine alpha may be employed to bind to andendokine alpha activity to treat ARDS, by preventing infiltration ofneutrophils into the lung after injury. The antagonists and antagonistsof the instant may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as described hereinafter.

Agonists and antagonists of the invention also have uses in stimulatingwound and tissue repair, stimulating angiogenesis, stimulating therepair of vascular or lymphatic diseases or disorders. Additionally,agonists and antagonists of the invention may be used to stimulate theregeneration of mucosal surfaces.

The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to. Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors tha may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Endokine alpha compositions of the invention are also suitablyadministered by sustained-release systems. Suitable examples ofsustained-release compositions include suitable polymeric materials(such as, for example, semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapules), suitable hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, and EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed.Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release compositions also include liposomally entrappedcompositions of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing Endokinealpha polypeptide may be prepared by methods known per se: DE 3,218,121;Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theEndokine alpha polypeptide therapy.

In another embodiment systained release compositions of the inventioninclude crystal formulations known in the art.

In yet an additional embodiment, the compositions of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

The compositions of the invention may be administered alone or incombination with other adjuvants. Adjuvants that may be administeredwith the compositions of the invention include, but are not limited to,alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, compositionsof the invention are administered in combination with alum. In anotherspecific embodiment, compositions of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe compositions of the invention include, but are not limited to,monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the compositions of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis, and/or PNEUMOVAX-23™. Combinations may beadministered either concomitantly, e.g., as an admixture, separately,but simultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately, but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

In another specific embodiment, compositions of the invention are usedin combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnoseinfection and/or any disease, disorder, and/or condition associatedtherewith. In one embodiment, compositions of the invention are used incombination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose anyGram positive bacterial infection and/or any disease, disorder, and/orcondition associated therewith. In another embodiment, compositions ofthe invention are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the genus Enterococcusand/or the genus Streptococcus. In another embodiment, compositions ofthe invention are used in any combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the Group Bstreptococci. In another embodiment, compositions of the invention areused in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with Streptococcus pneumoniae.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including, but not limitedto, chemotherapeutic agents, antibiotics, antivirals, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeutic agentsand cytokines. Combinations may be administered either concomitantly,e.g., as an admixture, separately, but simultaneously or concurrently;or sequentially. This includes presentations in which the combinedagents are administered together as a therapeutic mixture, and alsoprocedures in which the combined agents are administered separately, butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II(International Publication No. WO 97/34911 and WO 98/18921), APRIL (J.Exp. Med. 188(6):1185-1190), endokine-alpha (International PublicationNo. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG,OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30,CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095),DR3 (International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12.

In a preferred embodiment, the compositions of the invention areadministered alone or in combination with CD40 ligand (CD40L), a solubleform of CD40L (e.g., AVREND), biologically active fragments, variants,or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat, prevent, and/or diagnoseAIDS and/or to treat, prevent, and/or diagnose HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPR™-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/orETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose anopportunistic Mycobacterium avium complex infection. In another specificembodiment, compositions of the invention are used in any combinationwith RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticMycobacterium tuberculosis infection. In another specific embodiment,compositions of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat,prevent, and/or diagnose an opportunistic cytomegalovirus infection. Inanother specific embodiment, compositions of the invention are used inany combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™to prophylactically treat, prevent, and/or diagnose an opportunisticfungal infection. In another specific embodiment, compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic herpes simplex virus type I and/or type II infection. Inanother specific embodiment, compositions of the invention are used inany combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/ordiagnose an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs cyclophosphamide, cyclophosphamide IV, methylprednisolone,prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and otherimmunosuppressive agents that act by suppressing the function ofresponding T cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In a preferred embodiment, the compositions of the invention areadministered in combination with steroid therapy. Steroids that may beadministered in combination with the compositions of the invention,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, compositions of the invention are administered incombination with prednisone. In a further specific embodiment, thecompositions of the invention are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the compositions of the invention andprednisone are those described herein, and include, but are not limitedto, azathioprine, cylophosphamide, and cyclophosphamide IV. In a anotherspecific embodiment, compositions of the invention are administered incombination with methylprednisolone. In a further specific embodiment,the compositions of the invention are administered in combination withmethylprednisolone and an immunosuppressive agent. Immunosuppressiveagents that may be administered with the compositions of the inventionand methylprednisolone are those described herein, and include, but arenot limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the invention areadministered in combination with an NSAID.

In a nonexclusive embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, ten, ormore of the following drugs: NRD-101 (Hoechst Marion Roussel),diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin(Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000(Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-IRa genetherapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech),DW-166HC (Dong Wha), darbufelone mesylate (Wamer-Lambert), soluble TNFreceptor 1 (synergen; Amgen), IPR-6001 (Institute for PharmaceuticalResearch), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals),BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim),LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), andbutixocort propionate (WamerLambert).

In a preferred embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, and prednisolone.

In a more preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial, methotrexate, anti-TNFantibody, ENBREL™ and/or suflasalazine. In one embodiment, thecompositions of the invention are administered in combination withmethotrexate. In another embodiment, the compositions of the inventionare administered in combination with anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with methotrexate and anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with suflasalazine. In another embodiment, the compositionsof the invention are administered in combination with methotrexate,anti-TNF antibody, and suflasalazine. In another embodiment, thecompositions of the invention are administered in combination ENBREL™.In another embodiment, the compositions of the invention areadministered in combination with ENBREL™ and methotrexate. In anotherembodiment, the compositions of the invention are administered incombination with ENBREL™, methotrexate and suflasalazine. In anotherembodiment, the compositions of the invention are administered incombination with ENBREL™, methotrexate and suflasalazine. In otherembodiments, one or more antimalarials is combined with one of theabove-recited combinations. In a specific embodiment, the compositionsof the invention are administered in combination with an antimalarial(e.g., hydroxychloroquine), ENBREL™, methotrexate and suflasalazine. Inanother specific embodiment, the compositions of the invention areadministered in combination with an antimalarial (e.g.,hydroxychloroquine), sulfasalazine, anti-TNF antibody, and methotrexate.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but arenot limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, and IL-22. In preferred embodiments, the compositions ofthe invention are administered in combination with IL4 and IL10.

In an additional embodiment, the compositions of the invention areadministered with a chemokine. In another embodiment, the compositionsof the invention are administered with chemokine beta-8, chemokinebeta-1, and/or macrophage inflammatory protein-4. In a preferredembodiment, the compositions of the invention are administered withchemokine beta-8.

In an additional embodiment, the compositions of the invention areadministered in combination with an IL-4 antagonist. IL-4 antagoniststhat may be administered with the compositions of the invention include,but are not limited to: soluble IL-4 receptor polypeptides, multimericforms of soluble IL-4 receptor polypeptides; anti-IL-4 receptorantibodies that bind the IL-4 receptor without transducing thebiological signal elicited by IL-4, anti-IL4 antibodies that blockbinding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 thatbind IL-4 receptors but do not transduce the biological signal elicitedby IL-4. Preferably, the antibodies employed according to this methodare monoclonal antibodies (including antibody fragments, such as, forexample, those described herein).

In an additional embodiment, the compositions of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the compositions of the invention areadministered in combination with fibroblast growth factors. Fibroblastgrowth factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

Additionally, the compositions of the invention may be administeredalone or in combination with other therapeutic regimens, including, butnot limited to, radiation therapy. Such combinatorial therapy may beadministered sequentially and/or concomitantly.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the described disorders. Therapeutic compounds of theinvention include, but are not limited to, antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein). The antibodies of the invention can be used to treat, inhibitor prevent diseases, disorders or conditions associated with aberrantexpression and/or activity of a polypeptide of the invention, including,but not limited to, any one or more of the diseases, disorders, orconditions described herein such as, for example autoimmune diseases,disorders, or conditions associated with such diseases or disorders(including, but not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgAnephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis), systemic lupus erhythematosus,Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, forexample, Graves' Disease, Myasthenia Gravis, and insulin resistance,autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixedconnective tissue disease, polymyositis/dermatomyositis, perniciousanemia, idiopathic Addison's disease, infertility, glomerulonephritissuch as primary glomerulonephritis and IgA nephropathy, bullouspemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis), chronic active hepatitis, primary biliary cirrhosis, otherendocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomysyndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies,and other inflammatory, granulamatous, degenerative, and atrophicdisorders, and immunodeficiencies or conditions associated with suchdiseases or disorders, including, but not limited to, graft versus hostdisease and graft rejection.

In a specific embodiment, antibodies of the invention are be used totreat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, antibodies of the invention are used totreat, inhibit, prognose, diagnose or prevent systemic lupuserythematosis.

The treatment and/or prevention of diseases and disorders associatedwith aberrant expression and/or activity of a polypeptide of theinvention includes, but is not limited to, alleviating symptomsassociated with those diseases and disorders. Antibodies of theinvention may be provided in pharmaceutically acceptable compositions asknown in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M,10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁸M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, and 10⁻¹⁵ M.

In one embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing Endokine alpha polypeptides or anti-Endokinealpha antibodies associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs) to targeted cells, expressing themembrane-bound form of Endokine alpha on their surface, oralternativley, an Endokine alpha receptor (e.g., TR11) on their surface.Endokine alpha polypeptides or anti-Endokine alpha antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., Endokine alpha or anti-Endokinealpha antibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., Endokine alphapolypeptides or anti-Endokine alpha antibodies) in association withtoxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells expressing TR11 on their surface (e.g.,activated T cells, and/or T cell and/or B cell related leukemias orlymphomas) by administering Endokine alpha polypeptides in associationwith toxins or cytotoxic prodrugs.

In another specific embodiment, the invention provides a method for thespecific destruction of cells expressing the membrane-bound form ofEndokine alpha on their surface (e.g., endothelial cells) byadministering anti-Endokine alpha antibodies in association with toxinsor cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, cytotoxins (cytotoxic agents), or anymolecules or enzymes not normally present in or on the surface of a cellthat under defined conditions cause the cell's death. Toxins that may beused according to the methods of the invention include, but are notlimited to, radioisotopes known in the art, compounds such as, forexample, antibodies (or complement fixing containing portions thereof)that bind an inherent or induced endogenous cytotoxic effector system,thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” alsoincludes a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi,or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label proteins (includingantibodies) of the invention. Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety). A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is convertedby an enzyme, normally present in the cell, into a cytotoxic compound.Cytotoxic prodrugs that may be used according to the methods of theinvention include, but are not limited to, glutamyl derivatives ofbenzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubisin, andphenoxyacetamide derivatives of doxorubicin.

The compositions of the invention may be administered to an animal(including, but not limited to, those listed above, and also includingtransgenic animals) incapable of producing functional endogenousantibody molecules or having an otherwise compromised endogenous immunesystem, but which is capable of producing human immunoglobulin moleculesby means of a reconstituted or partially reconstituted immune systemfrom another animal (see, e.g., published PCT Application Nos. WO98/24893, WO 96/34096, WO 96/33735, and WO 91/10741). Compositions ofthe invention include, but are not limited to, endokine-alphapolypeptides and polynucleotides and agonists and antagonists thereof,antibodies, anti-antibodies, etc.

The compositions described herein may be used as a vaccine adjuvant thatenhances immune responsiveness to specific antigen. In a specificembodiment, the vaccine adjuvant is an endokine alpha polypeptidedescribed herein. In another specific embodiment, the vaccine adjuvantis an endokine alpha polynucleotide described herein (i.e., the endokinealpha polynucleotide is a genetic vaccine adjuvant). As discussedherein, endokine alpha polynucleotides may be administered usingtechniques known in the art, including but not limited to, liposomaldelivery, recombinant vector delivery, injection of naked DNA, and genegun delivery.

The compositions described herein may also be an adjuvant used toenhance tumor-specific immune responses.

Anti-viral immune responses that may be enhanced using the compositionsof the invention as an adjuvant, include, but are not limited to, virusand virus associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting of: AIDS,meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In anotherspecific embodiment, the compositions of the invention are used as anadjuvant to enhance an immune response to a virus, disease, or symptomselected from the group consisting of: HIV/AIDS, Respiratory syncytialvirus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B,Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya,Rift Valley fever, Herpes simplex, and yellow fever. In another specificembodiment, the compositions of the invention are used as an adjuvant toenhance an immune response to the HIV gp120 antigen.

Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of: tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria).

The compositions of the invention may be used as a stimulator of B or Tcell responsiveness to pathogens.

The compositions of the invention may be used as an agent that elevatesthe immune status of an individual prior to their receipt ofimmunosuppressive therapies; as an agent to induce higher affinityantibodies; as an agent to increase serum immunoglobulin concentrations;as an agent to accelerate recovery of immunocompromised individuals; asan agent to boost immunoresponsiveness among aged populations; and as animmune system enhancer prior to, during, or after bone marrow transplantand/or other transplants (e.g., allogeneic or xenogeneic organtransplantation).

With respect to transplantation, compositions of the invention may beadministered prior to, concomitant with, and/or after transplantation.In a specific embodiment, compositions of the invention are administeredafter transplantation, prior to the beginning of recovery of T-cellpopulations. In another specific embodiment, compositions of theinvention are first administered after transplantation after thebeginning of recovery of T cell populations, but prior to full recoveryof B cell populations.

The compositions of the invention may be used as an agent to boostimmunoresponsiveness among B cell and/or T cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy. B cell immunodeficiencies that may beameliorated or treated by administering the endokine alpha polypeptidesor polynucleotides of the invention, or agonists thereof, include, butare not limited to, severe combined immunodeficiency (SCID)-X linked,SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),X-linked agammaglobulinemia (XLA), Bruton's disease, congenitalagammaglobulinemia, X-linked infantile agammaglobulinemia, acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency. T cell immunodeficiencies that may beameliorated or treated by administering the endokine alpha polypeptidesor polynucleotides of the invention, or agonists thereof, include, butare not limited to, DiGeorge anomaly, thymic hypoplasia, chronicmucocutaneous candidiasis, natural killer cell deficiency, idiopathicCD4+ T-lymphocytopenia, and immunodeficiency with predominant T-celldefect, graft versus host disease, graft rejections and inflammationassociated with an immuno-deficiency.

Additional conditions resulting in an acquired loss of B or T cellfunction that may be ameliorated or treated by administering theendokine alpha polypeptides or polynucleotides of the invention, oragonists thereof, include, but are not limited to, HIV Infection, AIDS,bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

Compositions of the invention may also be used as an agent to boostimmunoresponsiveness among individuals having a temporary immunedeficiency. Conditions resulting in a temporary immune deficiency thatmay be ameliorated or treated by administering the endokine alphapolypeptides or polynucleotides of the invention, or agonists thereof,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, and recovery fromsurgery.

In a preferred embodiment, endokine alpha polynucleotides, polypeptides,and/or agonists and/or antagonists thereof are used to treat, prevent,and/or diagnose diseases or disorders affecting or conditions associatedwith any one or more of the various mucous membranes of the body. Suchdiseases or disorders include, but are not limited to, for example,mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (suchas, for example, American leishmaniasis, leishmaniasis americana,nasopharyngeal leishmaniasis, and New World leishmaniasis),mucocutaneous lymph node syndrome (for example, Kawasaki disease),mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor,mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration,myxoid degeneration, myxomatous degeneration, myxomatosis, mucoid medialdegeneration (for example, cystic medial necrosis), mucolipidosis(including, for example, mucolipidosis I, mucolipidosis II,mucolipidosis III, and mucolipidosis IV), mucolysis disorders,mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as,for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome),type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type Vmucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter'ssyndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome),type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VImucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VIImucopolysaccharidosis (i.e., mucopolysaccharidosis due tobeta-glucuronidase deficiency), and mucosulfatidosis),mucopolysacchariduria, mucopurulent conjunctivitis, mucopus,mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virusdiarrhea), mucous colitis (such as, for example, mucocolitis andmyxomembranous colitis), and mucoviscidosis (such as, for example,cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfieldsyndrome, fibrocystic disease of the pancreas, mucoviscidosis, andviscidosis). In a highly preferred embodiment, endokine alphapolynucleotides, polypeptides, and/or agonists and/or antagoniststhereof are used to treat, prevent, and/or diagnose mucositis,especially as associated with chemotherapy.

Endokine alpha polynucleotides or polypeptides of the invention, oragonists or antagonists thereof, may be used to diagnose, prognose,treat or prevent one or more of the following diseases or disorders, orconditions associated therewith: primary immuodeficiencies,immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrowtransplant (e.g., recent bone marrow transplant in adults or children),chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult orpediatric HIV infection), chronic inflammatory demyelinatingpolyneuropathy, and post-transfusion purpura.

Additionally, Endokine alpha polynucleotides or polypeptides of theinvention, or agonists or antagonists thereof, may be used to diagnose,prognose, treat or prevent one or more of the following diseases,disorders, or conditions associated therewith, Guillain-Barre syndrome,anemia (e.g., anemia associated with parvovirus B19), patients withstable mutliple myeloma who are at high risk for infection (e.g.,recurrent infection), autoimmune hemolytic anemia (e.g., warm-typeautoimmune hemolytic anemia), thrombocytopenia (e.g, neonatalthrombocytopenia), and immune-mediated neutropenia, transplantation(e.g, cytamegalovirus (CMV)-negative recipients of CMV-positive organs),hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with riskfactor for infection or morbidity), epilepsy (e.g, intractableepilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g,decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

Endokine alpha polynucleotides or polypeptides of the invention and/oragonists and/or antagonists thereof, may be used to treat, prevent,and/or diagnose various immune system-related disorders and/orconditions associated with these disorders, in mammals, preferablyhumans. Many autoimmune disorders result from inappropriate recognitionof self as foreign material by immune cells. This inappropriaterecognition results in an immune response leading to the destruction ofthe host tissue. Therefore, the administration of endokine alphapolynucleotides or polypeptides of the invention and/or agonists and/orantagonists thereof that can inhibit an immune response, particularlythe proliferation of B cells and/or the production of immunoglobulins,may be an effective therapy in treating and/or preventing autoimmunedisorders. Thus, in preferred embodiments, endokine alpha antagonists ofthe invention (e.g., polypeptide fragments of endokine alpha andanti-endokine alpha antibodies) are used to treat, prevent, and/ordiagnose an autoimmune disorder.

Autoimmune disorders and conditions associated with these disorders thatmay be treated, prevented, and/or diagnosed with the endokine alphapolynucleotides, polypeptides, and/or antagonists of the invention(e.g., anti-endokine alpha antibodies), include, but are not limited to,autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolyticanemia, antiphospholipid syndrome, dermatitis, allergicencephalomyelitis, myocarditis, relapsing polychondritis, rheumaticheart disease, glomerulonephritis (e.g, IgA nephropathy), MultipleSclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura(e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders (that are highly probable) that may betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to, autoimmune thyroiditis,hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized,e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupuserhythematosus (often characterized, e.g., by circulating and locallygenerated immune complexes), Goodpasture's syndrome (oftencharacterized, e.g., by anti-basement membrane antibodies), Pemphigus(often characterized, e.g., by epidermal acantholytic antibodies),Receptor autoimmunities such as, for example, Graves' Disease (oftencharacterized, e.g., by TSH receptor antibodies), Myasthenia Gravis(often characterized, e.g., by acetylcholine receptor antibodies), andinsulin resistance (often characterized, e.g., by insulin receptorantibodies), autoimmune hemolytic anemia (often characterized, e.g., byphagocytosis of antibody-sensitized RBCs), and autoimmunethrombocytopenic purpura (often characterized, e.g., by phagocytosis ofantibody-sensitized platelets.

Additional autoimmune disorders that may be treated, prevented, and/ordiagnosed with the compositions of the invention include, but are notlimited to, rheumatoid arthritis (often characterized, e.g., by immunecomplexes in joints), schleroderma with anti-collagen antibodies (oftencharacterized, e.g., by nucleolar and other nuclear antibodies), mixedconnective tissue disease (often characterized, e.g., by antibodies toextractable nuclear antigens (e.g., ribonucleoprotein)),polymyositis/dermatomyositis (often characterized, e.g., by nonhistoneANA), pernicious anemia (often characterized, e.g., by antiparietalcell, microsomes, and intrinsic factor antibodies), idiopathic Addison'sdisease (often characterized, e.g., by humoral and cell-mediated adrenalcytotoxicity, infertility (often characterized, e.g., byantispermatozoal antibodies), glomerulonephritis (often characterized,e.g., by glomerular basement membrane antibodies or immune complexes)such as primary glomerulonephritis and IgA nephropathy, bullouspemphigoid (often characterized, e.g., by IgG and complement in basementmembrane), Sjogren's syndrome (often characterized, e.g., by multipletissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetesmillitus (often characterized, e.g., by cell-mediated and humoral isletcell antibodies), and adrenergic drug resistance (including adrenergicdrug resistance with asthma or cystic fibrosis) (often characterized,e.g., by beta-adrenergic receptor antibodies).

Additional autoimmune disorders (that are possible) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, chronic active hepatitis (oftencharacterized, e.g., by smooth muscle antibodies), primary biliarycirrhosis (often characterized, e.g., by mitchondrial antibodies), otherendocrine gland failure (often characterized, e.g., by specific tissueantibodies in some cases), vitiligo (often characterized, e.g., bymelanocyte antibodies), vasculitis (often characterized, e.g., by Ig andcomplement in vessel walls and/or low serum complement), post-MI (oftencharacterized, e.g., by myocardial antibodies), cardiotomy syndrome(often characterized, e.g., by myocardial antibodies), urticaria (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), atopicdermatitis (often characterized, e.g., by IgG and IgM antibodies toIgE), asthma (often characterized, e.g., by IgG and IgM antibodies toIgE), inflammatory myopathies, and many other inflammatory,granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using anti-endokine alphaantibodies.

In a specific preferred embodiment, rheumatoid arthritis is treated,prevented, and/or diagnosed using anti-endokine alpha antibodies and/orother antagonist of the invention.

In a specific preferred embodiment, lupus is treated, prevented, and/ordiagnosed using anti-endokine alpha antibodies and/or other antagonistof the invention.

In a specific preferred embodiment, nephritis associated with lupus istreated, prevented, and/or diagnosed using anti-endokine alphaantibodies and/or other antagonist of the invention.

In a specific embodiment, endokine alpha polynucleotides orpolypeptides, or antagonists thereof (e.g., anti-endokine alphaantibodies) are used to treat or prevent systemic lupus erythematosusand/or diseases, disorders or conditions associated therewith.Lupus-associated diseases, disorders, or conditions that may be treatedor prevented with endokine alpha polynucleotides or polypeptides, orantagonists of the invention, include, but are not limited to,hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia,and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies,and anti-Sm antibodies), rashes, photosensitivity, oral ulcers,arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus(pleuricy)), renal disorders (e.g., nephritis), neurological disorders(e.g., seizures, peripheral neuropathy, CNS related disorders),gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In apreferred embodiment, the endokine alpha polynucleotides orpolypeptides, or antagonists thereof (e.g., anti-endokine alphaantibodies) are used to treat or prevent renal disorders associated withsystemic lupus erythematosus. In a most preferred embodiment, Endokinealpha polynucleotides or polypeptides, or antagonists thereof (e.g.,anti-endokine alpha antibodies) are used to treat or prevent nephritisassociated with systemic lupus erythematosus.

In certain embodiments, soluble endokine alpha polypeptides of theinvention, or agonists thereof, are administered, to treat, prevent,prognose and/or diagnose an immunodeficiency (e.g., severe combinedimmunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminasedeficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton'sdisease, congenital agammaglobulinemia, X-linked infantileagammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency, DiGeorge anomaly, thymic hypoplasia, chronicmucocutaneous candidiasis, natural killer cell deficiency, idiopathicCD4+ T-lymphocytopenia, and immunodeficiency with predominant T-celldefect or conditions associated with an immunodeficiency.

In a specific embodiment, endokine alpha polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, prognose and/or diagnose common variable immunodeficiency.

In a specific embodiment, endokine alpha polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, prognose and/or diagnose X-linked agammaglobulinemia.

In another specific embodiment, endokine alpha polypeptides orpolynucleotides of the invention, or agonists thereof, is administeredto treat, prevent, prognose and/or diagnose severe combinedimmunodeficiency (SCID).

In another specific embodiment, endokine alpha polypeptides orpolynucleotides of the invention, or agonists thereof, is administeredto treat, prevent, prognose and/or diagnose Wiskott-Aldrich syndrome.

In another specific embodiment, endokine alpha polypeptides orpolynucleotides of the invention, or agonists thereof, is administeredto treat, prevent, prognose and/or diagnose X-linked Ig deficiency withhyper IgM.

In another specific embodiment, endokine alpha polypeptides orpolynucleotides of the invention, or agonists thereof, is administeredto treat, prevent, prognose and/or diagnose DiGeorge anomaly.

In another specific embodiment, the combination of IL-10 and endokinealpha polypeptides or polynucleotides can advantageously be used in thesuppression of pathology, associated with T cell responses, inparticular, autoimmune diseases, graft-versus-host disease (GVHD) andtissue graft rejection. The invention can be used to suppresscell-mediated reactions such as allograft rejection and GVHD. Moreover,considering the diverse biological activities of IL-10, the concurrentuse of IL-10 and endokine alpha polypeptides or polynucleotides maysupport GVL (graft-versus-leukemia) in allogeneic bone marrowtransplants. In another specific embodiment, the compositions of theinvention may be used to prevent the rejection or prolong the survivalof allogeneic transplants of skin, bone, neuronal tissue, synovium,heart, kidney, pancreas, bone marrow, small intestine, lung, combinedheart-lung, corneal tissue, liver, etc. The transplanted tissue itselfis typically human in origin, but may also be from another species suchas a rhesus monkey, baboon or pig. As used herein, the term “tissue”includes individual cells, such as blood cells, including progenitorsand precursors thereof, and pancreatic cells, as well as solid organsand the like. The term solid organ means a heart, skin, a liver, a lung,a cornea, a kidney, a pancreas, an intestine, endocrine glands, abladder, skeletal muscles, etc. In another specific embodiment, thecompositions of the invention may be used to treat a large category ofdiseases, prior to and/or after onset thereof, such diseases including,but not being limited to, autoimmune diseases, including, but notlimited to, inflammatory conditions with an etiology including anautoimmune component such as arthritis (for example rheumatoidarthritis, arthritis chronica progrediente and arthritis deformans) andrheumatic diseases.

Autoimmune diseases may be divided into two general types, namelysystemic autoimmune diseases (exemplified by arthritis, lupus andscleroderma), and organ specific (exemplified by multiple sclerosis,diabetes and atherosclerosis, in which latter case the vasculature isregarded as a specific organ). Specific autoimmune diseases for whichthe compositions of the invention may be employed include, but, are notlimited to, autoimmune hematological disorders (including, e.g.,hemolytic anemia, aplastic anemia, pernicious anemia, pure red cellanemia and idiopathic thrombocytopenia), systemic lupus erythematosus,polychondritis, scleroderma, Wegener granulomatosis, dermatomyositis,chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnsonsyndrome, idiopathic sprue, autoimmune inflammatory bowel disease(including, e.g., ulcerative colitis and Crohn's disease), autoimmunethyroiditis, idiopathic Addison's disease, vitilogo, gluten-sensitiveenteropathy, autoimmune neutropenias, pemphigus vulgaris, Goodpasture'sdisease, bullous pemphigoid, discoid lupus, dense deposit disease,endocrine ophthalmopathy, IBD, asthma, Graves disease, sarcoidosis,multiple sclerosis, cirrhosis including primary biliary cirrhosis,juvenile diabetes (diabetes mellitus type 1), insulin dependent diabetesmellitus (i.e., IDDM, or autoimmune diabetes), uveitis (anterior andposterior), autoimmune gastritis, lymphopenias, polyarteritis nodosa,Sjogren's syndrome, Bechet's disease, Hashimoto's disease, primarymyxedema, polymyositis, mixed connective tissue disease,keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, psoriatic arthritis, glomerulonephritis (with and withoutnephrotic syndrome, e.g., including idiopathic nephrotic syndrome orminimal change nephropathy), juvenile dermatomyositis, hepatitisincluding chronic active hepatitis, organ rejection, and otherafflictions. Other diseases and conditions include, but are not limitedto, autoimmune thyroiditis, autoimmune hemolytic anemia, and contactsensitivity disease, which may, for example, be caused by plant matter,such as poison ivy. Other diseases and conditions include, but are notlimited to, suppressing chronic and acute monophasic EAE, HIV-relatedconditions, AIDS, SCIDS, adjuvant arthritis, a lymphatic malignancy orimmune disorder, and neurodegenerative diseases such as amyotrophiclateral sclerosis, Alzheimer's disease, Parkinson's disease and primarylateral sclerosis. Further, the inventive compositions can be used topromote wound healing and to treat infectious diseases.

The diseases set forth above, as referred to herein, include thoseexhibited by animal models for such diseases, such as, for examplenon-obese diabetic (NOD) mice for IDDM and experimental autoimmuneencephalomyelitis (EAE) mice for multiple sclerosis. Other conditionsinclude immune system-related miscarriage and inflammatory disorders.The discoveries of the present invention may also be applied to treatautoimmune diseases which manifest as infertility, includingendometriosis. Further, it is becoming increasingly apparent that manyvascular disorders, including atherosclerotic forms of such disorders,have an autoimmune component, and a number of patients with vasculardisease have circulating auto antibodies. In general, the compositionsof the invention are useful in immunomodulation, especiallyimmunosuppression, and in the treatment of leukemias characterized byover-proliferation of T-lymphocytes, including virally-inducedleukemias, e.g., HTLV-1-induced leukemia. An improvement or ameliorationin immune function can be assessed by observation of partial or totalrestoration of the ability to mount an immune response. In the case ofautoimmune disease, an improvement or amelioration can best be assessedby a significant reduction or disappearance of a clinical symptomassociated with inflammation caused by the autoimmune disease, forexample, joint pain or swelling or stiffness in rheumatoid arthritis;number of major attacks in chronic-relapsing multiple sclerosis;stabilization or improvement of motor function in chronic progressivemultiple sclerosis; intestinal inflammation in the case of Chron'sdisease; and serological measurements (such as antibody todouble-stranded DNA, complement components and circulating immunecomplexes), and number and severity of skin flare-ups or myalgras,arthralgia, leukopenia, or thrombocytopenia for systemic lupuserythematosus. The symptoms which can be used to monitor efficacy of aregimen in autoimmune disease are generally well-known in the art. Thecompositions of the invention can be applied to induce T cell toleranceto a variety of antigens. For example, T cell tolerance can be inducedto a soluble antigen (e.g., a soluble protein). T cells can be tolerizedto antigens involved in autoimmune diseases or disorders associated withabnormal immune responses. For example, in one embodiment, the antigenis an autoantigen. In another embodiment, the antigen is an allergan.Alternatively, T cells can be tolerized to antigens expressed on foreigncells. Accordingly, in yet other embodiments, the antigen is analloantigen or xenoantigen. Induction of T cell tolerance toalloantigens and xenoantigens is of particular use in transplantation,for example to inhibit rejection by a transplant recipient of a donorgraft, e.g., a tissue or organ graft or bone marrow transplant.Additionally, tolerization of donor T cells within a bone marrow graftis useful for inhibiting graft versus host disease.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); TIBTECH 11(5): 155-215 (May 1993)). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);Zijlstra et al., Nature 342:435-438 (1989)). In specific embodiments,the expressed antibody molecule is a single chain antibody;alternatively, the nucleic acid sequences include sequences encodingboth the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where they are expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering them so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.); and WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors have been to deleteretroviral sequences that are not necessary for packaging of the viralgenome and integration into host cell DNA. The nucleic acid sequencesencoding the antibody to be used in gene therapy are cloned into one ormore vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including, but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92 (1985)) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cells used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth.Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc.61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler, eds., Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327).

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball, eds., Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide of theinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. in vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of Endokine Alpha in E.coli

The DNA sequence encoding the endokine alpha protein in the depositedcDNA clone is amplified using PCR oligonucleotide primers specific tothe amino terminal sequences of the endokine alpha protein. Additionalnucleotides containing restriction sites to facilitate cloning are addedto the 5′ and 3′ sequences, respectively.

The 5′ oligonucleotide primer has the sequence GCG CCA TGG CTA AGT TTGGAC CAT (SEQ ID NO:5) containing the underlined Nco I restriction site.

The 3′ primer has the sequence GCG AAG CTT TCA AGT CTC TAG GAG ATG (SEQID NO:6) containing the underlined HindIII restriction site.

The restriction sites are convenient to restriction enzyme sites in thebacterial expression vector pQE60, which is used for bacterialexpression in M15/rep4 host cells in these examples. (Qiagen, Inc.,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Amp^(r)”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

The amplified endokine alpha protein DNA and the vector pQE60 both aredigested with NcoI and HindIII and the digested DNAs are then ligatedtogether. Insertion of the endokine alpha protein DNA into therestricted pQE60 vector places the endokine alpha protein coding regiondownstream of and operably linked to the vector's IPTG-induciblepromoter and in-frame with an initiating ATG appropriately positionedfor translation of endokine alpha protein.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressingendokine alpha protein, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 NM (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2×PBS.

Example 2 Cloning and Expression of Endokine Alpha in a BaculovirusExpression System

The cDNA sequence encoding the endokine alpha protein in the depositedclone is amplified using PCR oligonucleotide primers corresponding to 5′and 3′ regions of the gene.

The 5′ primer has the sequence GC GGA TCC CGA GAC TGC TAA GGA GCC (SEQID NO:7) containing the underlined BamHI restriction enzyme site andcontaining nucleotides encoding a portion of the endokine alpha proteinin FIG. 1.

The 3′ primer has the sequence GC GGA TCC CTA GGA GAT GAA TTG GGG ATT TG(SEQ ID NO:8) containing the underlined BamHI restriction site andcontaining a sequence complementary to that encoding a portion of theendokine alpha protein in FIG. 1.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and again is purifiedon a 1% agarose gel. This fragment is designated herein F2.

The vector pA2-GP is used to express the endokine alpha protein in thebaculovirus expression system, using standard methods, as described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites. The signalpeptide of AcMNPV gp67, including the N-terminal methionine, is locatedjust upstream of a BamHI site. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For an easyselection of recombinant virus, the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedrin promoter and isfollowed by the polyadenylation signal of the polyhedrin gene. Thepolyhedrin sequences are flanked at both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2-GP, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

The plasmid is digested with the restriction enzyme BamHI and then isdephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA is then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated herein “V2”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human endokine alpha gene by digesting DNA fromindividual colonies using BamHI and then analyzing the digestion productby gel electrophoresis. The sequence of the cloned fragment is confirmedby DNA sequencing.

5 μg of the plasmid is co-transfected with 1.0 μg of a commerciallyavailable linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”,Pharmingen, San Diego, Calif.), using the lipofection method describedby Felgner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 μgof BaculoGold™ virus DNA and 5 μg of the plasmid are mixed in a sterilewell of a microtiter plate containing 50 μl of serum-free Grace's medium(Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectinplus 90 μl Grace's medium are added, mixed and incubated for 15 minutesat room temperature. Then the transfection mixture is added drop-wise toSf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is rocked back andforth to mix the newly added solution. The plate is then incubated for 5hours at 27° C. After 5 hours the transfection solution is removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. The plate is put back into an incubator andcultivation is continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted endokine alpha is identified by DNAanalysis including restriction mapping and sequencing of this plasmid.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus at a multiplicity of infection (“MOI”) of about 2 (about 1to about 3). Six hours later the medium is removed and is replaced withSF900 II medium minus methionine and cysteine (available from LifeTechnologies Inc., Gaithersburg). 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3 Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of endokine alpha protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., J Biol. Chem.253:1357-1370 (1978), Hamlin, J. L. and Ma, C., Biochem. et Biophys.Acta, 1097:107-143 (1990), Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68) (1991). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human β-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express theendokine alpha in a regulated way in mammalian cells (Gossen, M., &Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well. Stable cell lines carryinga gene of interest integrated into the chromosomes can also be selectedupon co-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHI andAsp718I and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete endokine alpha protein includingits leader sequence is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene. The 5′ primer hasthe sequence 5′ GCG GGA TCC GCC ATC ATG CCT TTA AGC CAT TC 3′ (SEQ IDNO:9) containing the underlined BamHI restriction enzyme site followedby an efficient signal for initiation of translation in eukaryotes, asdescribed by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 17 basesof the coding sequence of endokine alpha shown in FIG. 1 (SEQ ID NO:1).The 3′ primer has the sequence 5′ GC GGA TCC CTA GGA GAT GAA TTG GGG ATTTG 3′ (SEQ ID NO:10) containing the underlined Asp718I restriction sitefollowed by nucleotides complementary to the non-translated region ofthe endokine alpha gene shown in FIG. 1 (SEQ ID NO:1).

The amplified fragment is digested with the endonucleases BamHI andAsp718I and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. 5 μg of the expression plasmid pC4 is cotransfected with0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al., supra).The plasmid pSV2neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 Tissue Distribution of Endokine Alpha Expression

Northern blot analysis was carried out to examine the levels ofexpression of the gene encoding the endokine alpha protein in humantissues, using methods described by, among others, Sambrook et al.,supra. A cDNA probe containing the entire nucleotide sequence of theendokine alpha protein of the present invention (SEQ ID NO:1) waslabeled with ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labelling, theprobe was purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labelled probe was then used to examine varioushuman tissues for the expression of the gene encoding the endokine alphaprotein.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) were obtained from Clontech andwere examined with labelled probe using ExpressHyb™ HybridizationSolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots were mountedand exposed to film at −70° C. overnight, and films developed accordingto standard procedures.

Expression of the gene encoding an endokine alpha protein of the presentinvention was detected in human brain striatum and pancreas tissue.

Example 5 Identification of a Novel Activation-Inducible Protein of theTNF Receptor Superfamily and its Ligand

Background

Members of the TNFR superfamily share similar multiple cysteine-richpseudorepeats of the extracellular domain, each containing 30-45 aminoacids with six cysteines (Smith, C. A., et al, Cell 76:959-962 (1994)).Except for the death domain-containing family which includes TNFR1(Schall, T. J., et al, Cell 61:361-370 (1990)), Fas (Trauth, B. C., etal, Science 245:301-305 (1989), Yonehara, S., et al, J. Exp. Med. 169:1747-1756 (1989), and Oehm, A., et al, J. Biol. Chem. 267:10709-10715(1992)), DR3 (Chinnaiyan, A. M., et al, Science 274:990-992 (1996),Kitson, J., et al, Nature 384:372-375 (1996), Bodmer, J.-L., et al,Immunity 6:79-88 (1997), and Screaton, G. R., et al, Proc. Natl. Acad.Sci. USA 94:4615-4619 (1997)), DR4 (Wiley, S. R., et al, Immunity3:673-682 (1995), Pitti, R. M., et al, J. Biol. Chem. 271:12687-2690(1996), and Pan, G., et al, Science 276:111-113 (1997)), DR5 (Walczak,H., et al, EMBO J. 16:5386-5397 (1997), MacFarlane, M., et al, J. Biol.Chem. 272:25417-25420 (1997), Schneider, P., et al, Immunity 7:831-836(1997), Chaudhary, P. M., et al, Immunity 7:821-830 (1997), andSheridan, J. P., et al, Science 277:818-821(1997)), and decoy TRAILreceptors (Marsters, S. A., et al, Cur. Biol. 7:1003-1006 (1997), Pan,G., et al, Science 277:815-815 (1997), Degli-Esposti, M. A., et al, J.Exp. Med. 186:1165-1170 (1997), and Degli-Esposti, M. A., et al,Immunity 7:813-820 (1997)), no remarkable similarity is found within theintracellular domain of these molecules. However, there is a strikinghomology in the cytoplasmic domains of murine and human 4-1BB, CD27, andmurine GITR within TNFR superfamily members (Kwon, B. S., et al, Proc.Natl. Acad. Sci. USA 86:1963-1967 (1989), Camerimi, D., et al, J.Immunol. 147:3165-3169 (1991), and Nocentini, G., et al, Proc. Natl.Acad. Sci. USA 94:6216-6221 (1997)). Acidic amino acids are especiallyhighly conserved in the cytoplasmic domain of this subfamily. Like otherTNFR superfamily members (Smith, C. A., et al, Cell 76:959-962 (1994)),this subfamily is implicated in diverse biological functions. First ofall, 4-1 BB and CD27 molecules provide strong costimulatory signals forT cell proliferation when ligated with their respective ligands or withagonistic antibodies (Smith, C. A., et al, Cell 76:959-962 (1994), andPollok, K. E., et al, J. Immunol. 150:771-781 (1993)). In addition tofunctioning as an accessory molecule, CD27 induces apoptosis, which ismediated by a death domain-containing molecule called Siva (Prasad, K.V. S., et al, Proc. Natl. Acad. Sci. USA 94:6346-6351 (1997)). Recentlyidentified murine GITR is shown to inhibit TCR-induced apoptosis(Nocentini, G., et al, Proc. Natl. Acad. Sci. USA 94:6216-6221 (1997)).

Although the immunological functions of subfamily members have beenrelatively well defined, insights into their signal transduction pathwayhave only recently been revealed (Arch, R. H., et al, Mol. Cell. Biol.18:558-565 (1998), Jang, I. K., et al, Biochem. Biophys. Res. Com.242:613-620 (1998), Saoulli, K., et al, J. Exp. Med. 187:1849-1862(1998), and Akiba, H., et al, J. Biol. Chem. 273:13353-13358 (1998)).Two groups (Arch, R. H., et al, Mol. Cell. Biol. 18:558-565 (1998), andJang, I. K., et al, Biochem. Biophys. Res. Com. 242:613-620 (1998)) haveprovided data indicating that association of 4-1BB with TRAF2 moleculesinitiates a signal cascade leading to activation of NF-κB. In the CD27signaling pathway, both TRAF2 and TRAF5 mediate NF-κB and SAPK/JNK(stress-activated protein kinase/c-Jun N-terminal kinase) activation andNIK (NF-κB-inducing kinase) is a common downstream kinase of TRAF2 andTRAF5 (Akiba, H., et al, J. Biol. Chem. 273:13353-13358 (1998)).

Because the number of TNFR members is rapidly expanding, it was expectedthat even more numbers of the superfamily would exist. By a PCR-basedstrategy with murine GITR sequence and searching an EST (expressedsequence tag) database, a new member of the TNFR was discovered andnamed TR11. The following provides a characterization of the receptorTR11 and its ligand, endokine alpha.

Experimental Procedures

cDNA cloning. A database containing more than two million ESTs obtainedfrom over 750 different cDNA libraries was generated by Human GenomeSciences, Inc., using high throughput automated DNA sequence analysis ofrandomly selected human cDNA clones. A specific homology and motifsearch using the known amino acid sequence and motif of TNFR membersagainst this database revealed several ESTs with a translated sequence35-55% homologous to that of the TNFR family. Several clones wereidentified from cDNA libraries of PHA-activated T cells, T helper cells,leukocytes, a healing abdomen wound, primary dendritic cells and adiposetissue. A full-length TR-11 cDNA clone encoding an intact N-terminalsignal peptide was obtained from a human activated T-cell library andselected for further investigation (see, U.S. patent application Ser.No. 09/176,200 filed Oct. 21, 1998). The complete cDNA sequence of bothstrands of this clone was determined, and its homology to TNFR memberswas confirmed. The same gene was also identified by a PCR-based strategywith murine GITR sequence. Similarly, endokine-α (TNF ligand 6) wasidentified through a systematic comparison of sequence homology with TNFligand family members. Partial endokine-α sequences which were 25%homologous to that of TNF ligand family members were identified fromendothelial, HUVEC (human umbilical vein endothelial cell), brain, andfetal liver cDNA libraries. A full-length cDNA clone was obtained from ahuman brain cDNA library.

Expression vectors. Full-length and HA (hemaglutinin A epitope)-taggedTR-11 encoding the putative entire TR-11 protein (amino acids 26-234)were amplified by PCR using sense (5′-CTAGCTAGCTAGVVVAGCGCCCCACCGGGGGTCCC-3′, and 5′-CTAGCTAGCTAGCTATCCATATGATGTTCCAGATTATGCTCAGCGCCCCACCGGGGGTCCC-3′, respectively) and anti-sense(5′-AAGGAAAAAAGCGGGCCGCTCA CACCCACAGGTCTCCCAG-3′) primers, cut with NheI/Not I, and fused in frame downstream of a CD5 leader sequence (Jang,I. K., et al, Biochem. Biophys. Res. Com. 242:613-620 (1998)) into thepcDNA3.1 (pcDNA3.1/CD5L-TR-11) and pcDNA3 (pcDNA3/CD5L-TR-11),respectively. Full-length endokine-α was amplified by PCR (sense,5′-AGACCCAAGCTTTTGAAAATGAT ATGAGACGC-3′; anti-sense,5′-AGACGGGATCCTCCTCCTATAGTAA GAAGGC-3′), cut with Hind III/BamH I, andinserted into pcDNA3.1 (pcDNA3.1/endokine-α) and pCEP4 (Invitrogen,Carlsbad, Calif.; pCEP4/endokine-α). pRK5-based expression vectorsencoding Flag-tagged full-length TRAF1, TRAF2, TRAF3, TRAF5, TRAF6, NIK,dominant negative TRAF2 (dnTRAF2), or dnNIK have been described (Jang,I. K., et al, Biochem. Biophys. Res. Com. 242:613-620 (1998), Rothe, M.,et al, Science 269:1421-1427 (1995), Hu, H. M., et al, J. Biol. Chem.269:30069-30072 (1994), Nakano, H., et al, J. Biol. Chem.271:14661-14664 (1996), Takeuchi, M., et al, J. Biol. Chem.271:19935-19942 (1996), Cao, Z., et al, Nature 383:443-446 (1996), andSong, H. Y., et al, Proc. Natl. Acad. Sci. USA 94:9792-9796 (1997)). TheNF-κB-dependent E-selectin-luciferase reporter gene (pELAM-Luc) andpRSV-β-galactosidase (pRSV-β-gal) plasmids were also described elsewhere(Rothe, M., et al, Science 269:1421-1427 (1995), and Schindler, U., etal, Mol Cell. Biol. 14:5820-9796 (1994)).

Northern blot and RT (reverse transcriptase)-PCR analysis. For Northernblot analysis, cDNA probes were labeled with ³²P using the Rediprime DNAlabeling system (Amersham Life Science, Arlington Height, Ill.),according to the manufacturer's instructions. Unincorporated nucleotidewas removed from the labeled probe using CHROMA SPIN-100 (Clonetech,Palo Alto, Calif.). Two human multiple tissue poly (A) RNA blotscontaining approximately 2 μg of poly (A) RNA per lane from varioushuman tissues were purchased from Clontech. In addition, two cell lineblots containing 20 mg total RNA from different cell lines were used.Northern blotting was performed with the Expressed HybridizationSolution (Clonetech) according to the manufacturer's manual. For RT-PCRanalysis, total RNA was isolated from human PBMC after stimulation withdexamethasone, PMA/ionomycin, or anti-CD3/CD28 mAbs, and fromunstimulated or LPS-stimulated HUVEC cells. RT-PCR was performed understandard conditions.

Interaction of TR-11 with TRAFs. pcDNA3/CD5L-TR-11-HA plasmid (5 μg/10cm-plate) was co-transfected into HEK293 EBNA cells (2×10⁶ cells/plate)by the standard calcium phosphate precipitation method with pRK/TRAF1,2, 3, 5, or 6-Flag vector (5 μg/plate). Twenty four-hours aftertransfection, cells were lysed with 1 ml of lysis buffer (50 mM HEPES[pH7.4], 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 10% glycerol, andprotease inhibitors). For immunoprecipitation, lysates were incubatedwith anti-Flag M2 (Eastman Kodak, Rochester, N.Y.) or control murineIgG1 mAb at 4° C. for 1 h, followed by incubation with 20 μl of a 1:1slurry of protein G-Sepharose (PharMingen, San Diego, Calif.) foranother hour. Precipitates were thoroughly washed with lysis buffer,then fractionated on a 10% SDS-polyacrylamide gel before transfer toPVDF membrane (Millipore, Bedfore, Mass.). Western blot analysis wasperformed with anti-HA mAb coupled with horseradish peroxidase(Boehringer Mannheim, Indianapolis, Ind.) and visualized using theenhanced chemiluminescence Western blotting detection system (Amersham).

Analysis of NF-κB by reporter assay. Approximately 0.5×10⁶ HEK293 EBNAcells/well were seeded on 6-well plates. After 24 h, cells weretransfected by the standard calcium phosphate precipitation method usingvarious combinations of pcDNA3.1/CD5L-TR-11 plus pRK5 plasmids encodingTRAFs, dnTRAF2, NIK, or dnNIK. The total amount of plasmid was adjustedto 2.0 μg by adding empty vector. Twenty-four hours after transfection,cells were lysed in 200 μl reporter lysis buffer (Promega, Madison,Wis.). Luciferase activity was measured using 20 μl cell extract. 5 μlcell extract was used to assay β-galactosidase activity as an internalcontrol, and luminescence values were normalized by individualβ-galactosidase activity.

Recombinant protein production and purification. TR-11-Fc fusion proteinwas used for ligand screening and cell-binding experiments. A fragmentencoding the predicted extracellular domain of TR-11 (amino acids26-139) was amplified using a sense primer flanked by an Nhe I site(5′-AGACCCAAGCTTGTGGGCTCTTGAAACCCGGCATG-3′) and an antisense primerflanked by a Bgl II site (5′-GAAAGATCTGGGCTCTGCCGG CGGGGACCCTGGGAC-3′).The amplified fragment was cut with Nhe I/Bgl II and cloned intomammalian vector pCEP4, in frame with CD5L at the 5′ end and with the Fcportion of human IgG1 at the 3′ end (pCEP4/CD5L-TR-11-Fc).pCEP4/CD5L-TR-11-Fc was transfected into HEK293 EBNA cells. TR-11-Fcfusion protein was purified from pCEP4/CD5L-TR-11-Fc-transfected HEK293EBNA cell supernatants using protein G column. To generate a Flag-taggedsoluble form of endokine-α protein (amino acids 39-169), the flag-taggedendokine-α expression vector (pCEP4/CD5L-endokine-α-Flag) wasconstructed by PCR amplification of endokine-α coding sequences usingsense(5′-CTAGCTAGCCCAGCGCCCCGACTACAAGGACGACGATGACAAGGAGACTGCTAAGGAGCCC-3′)and antisense (5′-CCGCTCGAGCTATAG TAAGAAGGCTCC-3′) primers, digestingthe product with Nhe I/Xho I and cloning into pCEP4, in frame with theCD5L sequence. The construct was expressed in HEK293 EBNA cells.Transfected cell supernatants containing secreted endokine-α-Flag wereharvested and used for binding assays. For some experiments,endokine-α-Flag protein was purified from harvested supernatants, usinganti-Flag gel (Sigma, St. Louis. MO) according to the manufacturer'sinstructions.

Binding assay. Protein binding assays were done essentially as described(Pan, G., et al, Science 276:111-113 (1997)). For cell-binding assays,HEK293 EBNA cells were transfected using pcDNA3.1/CD5L-TR-11 orpcDNA3.1, as described above. Forty-eight hours after transfection,cells were harvested and incubated consecutively withendokine-α-Flag-containing supernatant, anti-Flag antibody, andFITC-conjugated anti-mouse IgG antibody (Southern Biotechnology,Birmingham, Ala.). Flow cytometry analysis was performed using theBecton Dickinson FACScan (San Jose, Calif.). Jurkat T cells were stablytransfected by electroporation using linearized pcDNA3.1/CD5L-TR-11, andselected in the presence of Zeocin (Invitrogen). A binding assay forthis cell line was performed as described above. To test the ability ofTR-11-Fc fusion protein to bind membrane-bound endokine-α,pCEP4/endokine-α was stably transfected into HEK293 EBNA cells. Afterselection in the presence of hygromycin, endokine-α-expressing cellswere harvested and incubated with TR-11-Fc protein, followed byFITC-conjugated anti-human IgG1 antibody (Southern Biotechnology). TheBecton Dickinson FACScan was used for flow cytometry analysis.

Results and Discussion

TR-11 was identified by searching an EST database and by a PCR-basedstrategy with murine GITR sequence. A full-length cDNA of a clone from ahuman activated T-cell cDNA library, which is tentatively named TR-11(for activation-inducible TNFR family member), encodes a 234 amino acidtype I transmembrane protein with a calculated MW of 25 kDa. Thereceptor has a signal peptide (the first 25 amino acids) and a singletransmembrane region (amino acids 140-158). When compared with theextracellular domain of other TNFR family members, TR-11 displays threecysteine-rich pseudorepeats corresponding to the second, third, andfourth TNFR motif, respectively. The first cysteine pseudorepeatcontains eight cysteine residues and lacks C4. Therefore, it is unlikelythat the canonical pattern of C1-C2, C3-C5, and C4-C6 disulfide bridgesexist in this motif. The second pseudorepeat shows some features of thethird TNFR motif, but it is atypical in that C5 is not present eventhough it contains 7 cysteine residues. The third pseudorepeat showsextensive homologies with the fourth pseudorepeat of 4-1BB. Thecytoplasmic domain contains acidic amino acids which are highlyconserved in the cytoplasmic domains of 4-1BB, CD27, and GITR. Overall,TR-11 exhibits a high homology (55% identity) to murine GITR, but thereis a mismatch in the first cysteine-rich pseudorepeat between GITR andTR-11, because the first pseudorepeat of GITR corresponds to the firstTNFR cysteine-rich motif (Nocentini, G., et al, Proc. Natl. Acad. Sci.USA 94:6216-6221 (1997)).

The expression of TR-11 mRNA was investigated in multiple human tissuesby Northern blot hybridization. 1.25-kb mRNA was detected in lymph node,PBL, and, weakly, in spleen. We also tested a variety of tumor celllines for expression of TR-11 mRNA. 1.25-kb message was detected only inthe colorectal adenocarcinoma cell line, SW480, among the cell linestested. The expression of virtually all members of the TNFR superfamilyis enhanced by antigen stimulation/lymphocyte activation (Smith, C. A.,et al, Cell 76:959-962 (1994)). Consistent with this idea, TR-11expression was upregulated in PBMC after stimulation. No TR-11 messagewas detectable in unstimulated PBMC when we used a sensitive RT-PCRmethod. TR-11 expression was clearly induced within 24 h by typical PBMCstimulation such as treatment with PMA plus ionomycin or solubleanti-CD3 plus anti-CD28 mAbs. FACS analysis for TR-11 expression,however, showed that a small population of activated PBMC expressedTR-11 on the cell surface at 48 h after stimulation, suggesting that aprolonged period of stimulation is required for maximum expression ofTR-11 (BK, unpublished data). Expression of TR-11 was not induced bytreatment with dexamethasone. This property was different from that ofGITR (Nocentini, G., et al, Proc. Natl. Acad. Sci. USA 94:6216-6221(1997)).

Recently it has been shown that 4-1BB molecules associate with TRAF1,TRAF2, and TRAF3 (Arch, R. H., et al, Mol. Cell Biol. 18:558-565 (1998),Jang, I. K., et al, Biochem. Biophys. Res. Com. 242:613-620 (1998), andSaoulli, K., et al, J. Exp. Med. 187:1849-1862 (1998)). Because TR-11'scytoplasmic domain is similar to that of 4-1BB, its ability toco-precipitate five of the six known TRAFs that were overexpressed inHEK293 EBNA cells was tested. An interaction of TR-11 with TRAF1, TRAF2,and TRAF3 was observed but not with TRAF5 and TRAF6. The association ofTR-11 with TRAF2 suggested that, like other members of the TNFRsuperfamily (Arch, R. H., et al, Mol Cell Biol. 18:558-565 (1998), Jang,I. K., et al, Biochem. Biophys. Res. Com. 242:613-620 (1998), Akiba, H.,et al, J. Biol. Chem. 273:13353-13358 (1998), Rothe, M., et al, Science269:1421-1427 (1995), Cheng, G., et al, Science 267:1494-1498 (1995),Ducke, C. S., et al, Mol Cell. Biol. 17:1535-1542 (1997), andVanArsdale, T. L., et al, Proc. Natl. Acad. Sci. USA 94:2460-2465(1996)), TR-11 might mediate NF-κB activation through TRAF2. To testthis possibility, an NF-κB reporter system in HEK293 EBNA cells was used(Rothe, M., et al, Science 269:1421-1427 (1995)). Co-transfection withthe TR-11 expression vector typically induced greater than 3-fold higherluciferase activity when compared with the vector transfection control.When co-expressed with TRAF2, TR-11 induced greater luciferase activitythan did TRAF2 alone. More importantly, overexpression ofdominant-negative TRAF2, which lacked the RING and zinc finger motifs(Rothe, M., et al, Science 269:1421-1427 (1995)), abrogated theluciferase activity induced by TR-11. This indicates that TRAF2 is animportant mediator of NF-κB activation for TR-11. A similar observationwas made when the activity of NIK, which was thought to lie downstreamof TRAF2 in the NF-κB signaling pathway, was blocked by overexpressionof the dominant-negative NIK (Song, H. Y., et al., Proc. Natl. Acad.Sci. USA 94:9792-9796 (1997)), which lacked the two lysine residues ofcatalytic domain. Taken together, these data indicate that TR-11mediates NF-κB activation through the TRAF2/NIK pathway. Since TRAF1 andTRAF3 were found to associate with TR-11 in HEK293 EBNA cells, theeffects of TRAF1 and TRAF3 on NF-κB activation induced by TR-11 wasexamined. The introduction of TRAF3 nearly abolished the luciferaseactivity induced by TR-11 overexpression. To a lesser extent, TRAF1overexpression diminished TR-11-induced NF-κB activation. These datasuggest that TRAF1 and especially TRAF3 downregulate TR-11-induced NF-κBactivation.

To identify TR-11 ligand, a panel of Flag-tagged candidate TNF ligandproteins for binding to TR-11-Fc fusion protein was screened byimmunoprecipitation. TR-11-Fc selectively bound endokine-α-Flag amongFlag-tagged TNF ligand proteins tested. In our experimental conditions,4-1BB and TR2 (HVEM) bound their cognate ligands, 4-1BBL and LIGHT(Mauri, D. N., et al, Immunity 8:21-30 (1998)), respectively.Furthermore, this data clearly showed that endokine-α-Flag protein boundTR-11 transiently expressed on the cell surface of HEK293 EBNA cells andTR-11 constitutively expressed on the cell surface of Jurkat cell. Sinceendokine-α is a transmembrane protein (see below), flow cytometry to wasused determine whether TR-11-Fc fusion protein was able to bind HEK293EBNA cells that were stably transfected with full length endokine-α. Theresults demonstrate that TR-11-Fc protein was capable of bindingendokine-α expressed on HEK293 EBNA cells.

Next, it was determined whether interactions between TR-11 andendokine-α would result in NF-κB activation. In an NF-κB reporter assay,ligand-dependent NF-κB activation was demonstrated by cotransfectingtransmembrane endokine-α with TR-11 or transfectingendokine-α-expressing HEK293 EBNA cells. In addition, when TR-11 wastransiently transfected into HEK293 EBNA cells which constitutivelysecreted soluble endokine-α protein, NF-κB activation markedly increasedas compared to empty vector-transfected HEK293 EBNA cells. Similarly,higher NF-κB activation was induced by treating with soluble endokine-αprotein HEK293 cells which were transiently transfected with TR-11. Thisindicates that endokine-α is able to trigger TR-11-specific activationof NF-κB. It appears that higher induction of NF-κB by endokine-α iscorrelated with a stronger association of TR-11 with TRAF2 in HEK293EBNA cells, since stronger association of TR-11 with TRAF2 was observedin cells which were cotransfected with endokine-α than in cells whichwere transfected with TR-11 alone.

Endokine-α was one of the TNF ligand proteins initially identified by anEST database search. Hydrophilicity analysis of a full-length endokine-αclone from a brain cDNA library predicts a single hydrophobictransmembrane domain and the absence of a signal sequence. Endokine-αcontains two potential glycosylation sites in the C-terminal region.These features suggest that endokine-α is a type II membrane proteinwith the C-terminal region extracellular. Northern blot analysis ofhuman tissue RNAs revealed expression of a single 2.4-kb endokine-α mRNAin pancreas. Various human cell lines and PBMC were also examined forendokine-α expression. No message was detectable in either unstimulatedor stimulated T-cell lines (CEM-6 and Jurkat), B-cell lines (Priess andFrev), promyelocytic cell line (HL-60), monocytic cell line (THP-1), andPBMC by RT-PCR. In contrast, HUVEC cells constitutively expressedendokine-α and its expression was upregulated after stimulation withLPS. Therefore, it is believed that TR-11 and its ligand are importantfor interactions between activated T lymphocytes and blood vessels.

TR-11 has 55% identity with murine GITR at the amino acid level. Thehigh sequence conservation between human and mouse provides evidencethat TR-11 is the human homologue of murine GITR. At this point,however, the possibility remains that these two receptors may servedistinct functions from one another, based on the following facts: (1)There is a mismatch in the first cysteine-rich pseudorepeat between GITRand TR-11; (2) in contrast to GITR, TR-11 is not inducible bydexamethasone.

In summary, a novel protein of the TNFR superfamily, TR-11, whichactivates NF-κB through a TRAF2-mediated mechanism has identified.Expression of TR-11 is activation-inducible. The ligand for TR-11,endokine-α, is a member of the TNF ligand family and is constitutivelyexpressed in an endothelial cell line. This indicates that TR-11 and itsligand may be involved in activated T-cell trafficking.

Example 6 The Effects of Endokine Alpha on Monocytes

These studies disclose that treatment with endokine-α induced TNF-α,MCP-1, IL-8 and IL-10 release from monocytes and inhibited theproduction of IL-12 in monocytes. (data not shown).

Methods

Monocyte purification. Peripheral blood mononuclear cells (PBMC) werepurified from single donor leukopacks (American Red Cross, Baltimore,Md.) by centrifugation through a Histopaque gradient (Sigma). Monocyteswere isolated from PBMC by counterflow centrifugal elutriation.

ELISA. Human monocytes were incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of endokine-α. For IL-12 production, thecells were primed overnight with IFN-γ (100 U/ml) in presence ofEndokine-α. LPS (10 ng/ml) was then added. Conditioned media wascollected after 24 h and kept frozen until use. ELISA kits for themeasurement of TNF-α, IL-10, MCP-1 and IL-8 were purchased from R & DSystems (Minneapolis, Minn.). Each value was the mean of triplicatesamples±standard deviation.

Oxidative burst. Purified monocytes were plated in 96-well plate at2-1×10⁵ cell/well. Increasing concentrations of Endokine-α are added tothe wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS,glutamine and antibiotics). After 3 days incubation, the plates arecentrifuged and the medium is removed from the wells. To the macrophagemonolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mMpotassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol redand 19 U/ml of HRPO) was added, together with the stimulant (200 nMPMA). The plates were incubated at 37° C. for 2 hours and the reactionwas stopped by adding 20 μl 1N NaOH per well. The absorbance was read at610 nm. To calculate the amount of H₂O₂ produced by the macrophages, astandard curve of a H₂O₂ solution of known molarity was done for eachexperiment.

Effect of Endokine-a Treatment on IL-12 Secretion by Monocytes

Treatment IL-12 Inhibition (mg/ml) (pg/ml) % — 513 TL-6 0.2 600 0 TL-6 1421 28 TL-6 5 54 89

Monocytes (5×10⁵/ml) were incubated with IFN-g (100 U/ml) and TL-6.After 16 hours, LPS (10 ng/ml) was added to the cultures. Conditionedmedia was collected 24 hours following LPS addition and analyzed inELISA for IL-12 content.

Example 7 Assays to Detect Stimulation or Inhibition of B CellProliferation and Differentiation Background

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL5, IL6,IL-7, IL10, IL-13, IL14 and IL15. Interestingly, these signals are bythemselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

Experimental Procedure

In vitro assay. Purified Endokine-α protein, or truncated forms thereof,is assessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of Endokine-α protein on purified humantonsillar B cells, measured qualitatively over the dose range from 0.1to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with ³H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In vivo assay. BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of endokine-α protein, or truncated forms thereof. Micereceive this treatment for 4 consecutive days, at which time they aresacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and endokine-α protein-treatedspleens identify the results of the activity of endokine-α protein onspleen cells, such as the diffusion of peri-arterial lymphatic sheaths,and/or significant increases in the nucleated cellularity of the redpulp regions, which may indicate the activation of the differentiationand proliferation of B-cell populations. Immunohistochemical studiesusing a B cell marker, anti-CD45R(B220), are used to determine whetherany physiological changes to splenic cells, such as splenicdisorganization, are due to increased B-cell representation withinloosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from endokine-α protein-treatedmice is used to indicate whether endokine-α protein specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andEndokine-α protein-treated mice.

Example 8 Assays to Detect Stimulation or Inhibition of T CellProliferation and Differentiation

The anti-CD3 and/or PHA costimulation assay is used to detected thestimulation or inhibition of T cell proliferation and differentiation.

Assay Parameters

Cells:

-   PBMC per well: 10⁵-   PBMC recovered per donor: 200×10⁶-   Total plates per day: 20-   Supematants per plate: 48 (each assayed in duplicate)-   Total supernatants per day per donor: 960 (two donors per day)-   Need an additional 4 units of blood/week to accommodate new assay.

Reagents:

-   anti-human CD3 mAb (25 pg/mL final concentration in each well)-   PHA-   rhIL-2 (positive control)-   ³H-thymidine (0.5 μCi/well, 6.7 Ci/mmole)-   96-well plates

Protocol:

-   Purify PBMC.-   Prepare plates with appropriate controls.-   Incubate at 37° C. for 3-4 days.-   Add ³H-TdR and return to incubator for an additional 20-24 hours.-   Harvest and count.    Outcomes

This assay allows the determination of whether Endokine-α enhances orinhibits anti-CD3-dependent proliferation of PBMCs and whetherEndokine-α stimulates PBMC proliferation in the absence of costimulatorysignals.

Example 9 Isolation of Antibody Fragments Directed Against Polypeptidesof the Present Invention from a Library of scFvs

Naturally occuring V-genes isolated from human PBLs are constructed intoa large library of antibody fragments which contain reactivities againstpolypeptides of the present invention to which the donor may or may nothave been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated hereinin its entirety by reference).

Rescue of the library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in WO92/01047. To rescue phage displayingantibody fragments, approximately 10⁹ E. coli harbouring the phagemidare used to inoculate 50 ml of 2×TY containing 1% glucose and 100 ug/mlof ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU.Next 2×10⁸ TU of delta gene 3 helper phage (M13 delta gene III, see WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 minutes and the pelletresuspended in 2 liters of 2×TY containing 100 ug/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inWO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harbouring a pUC19 derivative supplying the wild typegene III protein during phage morphogenesis. The culture is incubatedfor 1 hour at 37° C. without shaking and then for a further hour at 37°C. with shaking. Cells are pelleted (IEC-Centra 8,4000 revs/min for 10min), resuspended in 300 ml 2×TY broth containing 100 ug ampicillin/mland 25 ug kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at37° C. Phage particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 1990), resuspended in2 ml PBS and passed through a 0.45 um filter (Minisart NML; Sartorius)to give a final concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 mg/ml or 10 mg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 10¹³ TU of phageare applied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0 MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100ug/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (seee.g., WO92/01047) and then by sequencing.

Example 10 Method of Determining Alterations in the Endokine Alpha Gene

RNA is isolated from entire families or individual patients presentingwith a phenotype of interest (such as a disease). cDNA is then generatedfrom these RNA samples using protocols known in the art. (see,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 secondsat 52-58° C.; and 60-120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase(Epicentre Technologies). The intron-exon borders of selected exons ofendokine alpha are also determined and genomic PCR products analyzed toconfirm the results. PCR products harboring suspected mutations inendokine alpha are then cloned and sequenced to validate the results ofthe direct sequencing.

PCR products of endokine alpha are cloned into T-tailed vectors asdescribed in Holton, T. A. and Graham, M. W., Nucleic Acids Research,19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations inendokine alpha not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in the endokine alpha gene. Genomic clones isolated usingtechniques known in the art are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, C. G. et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to theendokine alpha genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, C. V. et al., Genet. Anal.Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of endokine alpha (hybridized by theprobe) are identified as insertions, deletions, and translocations.These endokine alpha alterations are used as a diagnostic marker for anassociated disease.

Example 11 Method of Detecting Abnormal Levels of Endokine Alpha in aBiological Sample

Endokine alpha polypeptides can be detected in a biological sample, andif an increased or decreased level of endokine alpha is detected, thispolypeptide is a marker for a particular phenotype. Methods of detectionare numerous, and thus, it is understood that one skilled in the art canmodify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect endokine alphain a sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies to endokine alpha, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced using technique known in the art. Thewells are blocked so that non-specific binding of endokine alpha to thewell is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining endokine alpha. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded endokinealpha.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Seventy-five ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution is then added to each well andincubated 1 hour at room temperature to allow cleavage of the substrateand flourescence. The flourescence is measured by a microtiter platereader. A standard curve is preparded using the experimental resultsfrom serial dilutions of a control sample with the sample concentrationplotted on the X-axis (log scale) and fluorescence or absorbance on theY-axis (linear scale). The endokine alpha polypeptide concentration in asample is then interpolated using the standard curve based on themeasured flourescence of that sample.

Example 12 Method of Treating Decreased Levels of Endokine Alpha

The present invention also relates to a method for treating anindividual in need of an increased level of endokine alpha biologicalactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of endokinealpha or an agonist thereof.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of endokine alpha in anindividual can be treated by administering endokine alpha, preferably ina soluble and/or secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofendokine alpha polypeptide comprising administering to such anindividual a pharmaceutical composition comprising an amount of endokinealpha to increase the biological activity level of endokine alpha insuch an individual.

For example, a patient with decreased levels of endokine alphapolypeptide receives a daily dose 0.1-100 μg/kg of the polypeptide forsix consecutive days. Preferably, the polypeptide is in a soluble and/orsecreted form.

Example 13 Method of Treating Increased Levels of Endokine Alpha

The present invention relates to a method for treating an individual inneed of a decreased level of endokine alpha biological activity in thebody comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of endokine alphaantagonist. Preferred antagonists for use in the present invention areendokine alpha-specific antibodies or endokine alpha antisensepolynucleotides.

Antisense technology is used to inhibit production of endokine alpha.This technology is one example of a method of decreasing levels ofendokine alpha polypeptide, preferably a soluble and/or secreted form,due to a variety of etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels ofendokine alpha is administered intravenously antisense polynucleotidesat 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the is determined to be welltolerated.

Example 14 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature endokine alpha polypeptides, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask, approximately ten pieces areplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted; the chunks of tissue remain fixed to the bottomof the flask and fresh media (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin) is added. The flasks are then incubated at37° C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding endokine alpha can be amplified using PCR primerswhich correspond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector contains properly inserted endokine alpha.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the endokine alpha gene is then added to the media and thepackaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the endokine alpha gene(the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether endokinealpha protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 15 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) endokine alpha sequences into an animal toincrease or decrease the expression of the endokine alpha polypeptide.The endokine alpha polynucleotide may be operatively linked to apromoter or any other genetic elements necessary for the expression ofthe endokine alpha polypeptide by the target tissue. Such gene therapyand delivery techniques and methods are known in the art, see, forexample, WO 90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151,5,580,859; Tabata H. et al., Cardiovasc. Res. 35:470-479 (1997); Chao J.et al., Pharmacol. Res. 35:517-522 (1997); Wolff J. A. Neuromuscul.Disord. 7:314-318 (1997); Schwartz B. et al., Gene Ther. 3:405-411(1996); Tsurumi Y. et al., Circulation 94:3281-3290 (1996) (incorporatedherein by reference).

The endokine alpha polynucleotide constructs may be delivered by anymethod that delivers injectable materials to the cells of an animal,such as, injection into the interstitial space of tissues (heart,muscle, skin, lung, liver, intestine and the like). The endokine alphapolynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the endokine alpha polynucleotides may also bedelivered in liposome formulations (such as those taught in Felgner P.L. et al. Ann. NY Acad. Sci. 772:126-139 (1995), and Abdallah B. et al.Biol. Cell 85:1-7 (1995)) which can be prepared by methods well known tothose skilled in the art.

The endokine alpha polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Any strong promoter known to those skilled in the art canbe used for driving the expression of DNA. Unlike other gene therapytechniques, one major advantage of introducing naked nucleic acidsequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The endokine alpha polynucleotide construct can be delivered to theinterstitial space of tissues within an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked endokine alpha polynucleotide injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05μg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration. The preferred route of administration is by theparenteral route of injection into the interstitial space of tissues.However, other parenteral routes may also be used, such as, inhalationof an aerosol formulation particularly for delivery to lungs orbronchial tissues, throat or mucous membranes of the nose. In addition,naked endokine alpha polynucleotide constructs can be delivered toarteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected endokine alpha polynucleotide inmuscle in vivo are determined as follows. Suitable endokine alphatemplate DNA for production of mRNA coding for endokine alphapolypeptide is prepared in accordance with a standard recombinant DNAmethodology. The template DNA, which may be either circular or linear,is either used as naked DNA or complexed with liposomes. The quadricepsmuscles of mice are then injected with various amounts of the templateDNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The endokine alpha template DNA is injected in 0.1 ml ofcarrier in a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 μmcross-section of the individual quadriceps muscles is histochemicallystained for endokine alpha protein. A time course for endokine alphaprotein expression may be done in a similar fashion except thatquadriceps from different mice are harvested at different times.Persistence of endokine alpha DNA in muscle following injection may bedetermined by Southern blot analysis after preparing total cellular DNAand HIRT supernatants from injected and control mice. The results of theabove experimentation in mice can be use to extrapolate proper dosagesand other treatment parameters in humans and other animals usingendokine alpha naked DNA.

Example 16 Gene Therapy Using Endogenous Endokine Alpha Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous endokine alpha sequencewith a promoter via homologous recombination as described, for example,in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication Number WO 96/29411, published Sep. 26, 1996; InternationalPublication Number WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989). This method involves the activation of a genewhich is present in the target cells, but which is not expressed in thecells, or is expressed at a lower level than desired. Polynucleotideconstructs are made which contain a promoter and targeting sequences,which are homologous to the 5′ non-coding sequence of endogenousendokine alpha, flanking the promoter. The targeting sequence will besufficiently near the 5′ end of endokine alpha so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousendokine alpha sequence. This results in the expression of endokinealpha in the cell. Expression may be detected by immunological staining,or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells are againcentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the endokine alpha locus,plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII.The CMV promoter is amplified by PCR with an XbaI site on the 5′ end anda BamHI site on the 3′end. Two endokine alpha non-coding sequences areamplified via PCR; one endokine alpha non-coding sequence (endokinealpha fragment 1) is amplified with a HindIII site at the 5′ end and anXba site at the 3′end; the other endokine alpha non-coding sequence(endokine alpha fragment 2) is amplified with a BamHI site at the 5′endand a HindIII site at the 3′end. The CMV promoter and endokine alphafragments are digested with the appropriate enzymes (CMV promoter—XbaIand BamHI; endokine alpha fragment 1—XbaI; endokine alpha fragment2—BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1-5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 msec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37_C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and the cells are incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 17 Effect of Endokine Alpha on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of endokine alpha or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (106/ml) are treated with increasing concentrations ofendokine alpha for 24 hours. LPS (100 ng/ml) is added to the cellculture as positive control. Supernatants from the cell cultures arethen collected and analyzed for IL-12 content using commercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)). The standard protocolsprovided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofendokine alpha or LPS (positive control), washed with PBS containing 1%BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4° C. After an additional wash, the labeled cells are analyzed by flowcytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Endokine alpha, agonists, or antagonists of endokine alphacan be screened using the three assays described below. For each ofthese assays, peripheral blood mononuclear cells (PBMC) are purifiedfrom single donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

1. Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from an internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha, dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2×10⁶/ml in PBS containing PI at a finalconcentration of 5 μg/ml, and then incubated at room temperature for 5minutes before FAC Scan analysis. PI uptake has been demonstrated tocorrelate with DNA fragmentation in this experimental paradigm.

2. Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of endokine alpha or in the absence ofendokine alpha. For IL-12 production, the cells are primed overnightwith IFN-γ (100 U/ml) in presence of endokine alpha. LPS (10 ng/ml) isthen added. Conditioned media is collected after 24 h and kept frozenuntil use. Measurement of TNF-α, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g., R & D Systems(Minneapolis, Minn.)) applying the standard protocols provided with thekit.

3. Oxidative burst. Purified monocytes are plated in 96-well plates at2-1×10⁵ cell/well. Increasing concentrations of endokine alpha are addedto the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10%FCS, glutamine and antibiotics). After 3 days incubation, the plates arecentrifuged and the medium is removed from the wells. To the macrophagemonolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mMpotassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol redand 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA).The plates are incubated at 37° C. for 2 hours and the reaction isstopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610nm. To calculate the amount of H₂O₂ produced by the macrophages, astandard curve of a H₂O₂ solution of known molarity is performed foreach experiment.

The studies described in this example tested activity in endokinealphaprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of endokine alphapolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofendokine alpha.

Example 18 Assay to Detect Stimulation or Inhibition of T CellProliferation

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100_l/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4_C(1_g/ml in 0.05 M bicarbonate buffer, pH 9.5), then washed three timeswith PBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of endokine alpha protein (total volume 200_l).Relevant protein buffer and medium alone are controls. After 48 hourculture at 37_C, plates are spun for 2 min. at 1000 rpm and 100_l ofsupernatant is removed and stored −20_C for measurement of IL-2 (orother cytokines) if effect on proliferation is observed. Wells aresupplemented with 100_l of medium containing 0.5_Ci of ³H-thymidine andcultured at 37_C for 18-24 hr. Wells are harvested and incorporation of³H-thymidine used as a measure of proliferation. Anti-CD3 alone is thepositive control for proliferation. IL-2 (100 U/ml) is also used as acontrol which enhances proliferation. Control antibody which does notinduce proliferation of T cells is used as the negative controls for theeffects of endokine alpha proteins.

The studies described in this example tested activity in endokine alphaprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of endokine alphapolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofendokine alpha.

Example 19 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (see, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing Endokine alpha are administered to an animalto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of Endokine alpha protein is preparedand purified to render it substantially free of natural contaminants.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

Monoclonal antibodies specific for protein Endokine alpha are preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monocloal/Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, ananimal preferably a mouse) is immunized with Endokine alpha polypeptideor, more preferably, with a secreted Endokine alphapolypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the Endokine alphapolypeptide.

Alternatively, additional antibodies capable of binding to Endokinealpha polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theEndokine alpha protein-specific antibody can be blocked by Endokinealpha. Such antibodies comprise anti-idiotypic antibodies to theEndokine alpha protein-specific antibody and are used to immunize ananimal to induce formation of further Endokine alpha protein-specificantibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (see, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

b) Isolation of Antibody Fragments Directed Against Endokine Alpha froma Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstEndokine alpha to which the donor may or may not have been exposed (seee.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 μg/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al, 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The disclosures of all patents, patent applications, and publicationsreferred to herein are hereby incorporated by reference.

1. A method of inhibiting an inflammatory immune response in a subject,comprising administering to the subject an effective amount of anisolated antibody or an antigen-binding fragment thereof that binds to aprotein consisting of an amino acid sequence selected from the groupconsisting of: (a) amino acids 1 to 169 of SEQ ID NO:2; (b) amino acids2 to 169 of SEQ ID NO:2; (c) amino acids 1 to 17 of SEQ ID NO:2; (d)amino acids 18 to 43 of SEQ ID NO:2; (e) amino acids 44 to 169 of SEQ IDNO:2; (f) amino acids 44 to 158 of SEQ ID NO:2; (g) amino acids 44 to 54of SEQ ID NO:2; (h) amino acids 57 to 68 of SEQ ID NO:2; (i) amino acids69 to 78 of SEQ ID NO:2; (j) amino acids 94 to 105 of SEQ ID NO:2; (k)amino acids 108 to 132 of SEQ ID NO:2; (l) amino acids 148 to 158 of SEQID NO:2; (m) at least 30 contiguous amino acids of SEQ ID NO:2; (n) thecomplete endokine alpha amino acid sequence encoded by the cDNA clone inATCC Deposit No. 97640; (o) the complete endokine alpha amino acidsequence encoded by the cDNA clone in ATCC Deposit No. 97640, minus theN-terminal methionine residue; (p) the extracellular domain of theendokine alpha encoded by the cDNA clone in ATCC Deposit No. 97640; and(q) at least 30 contiguous amino acids of the endokine alpha encoded bythe cDNA clone in ATCC Deposit No. 97640; wherein said antibody inhibitsTNF-α induction.
 2. The method of claim 1, wherein said antibody or anantigen-binding fragment thereof binds a protein consisting of (a). 3.The method of claim 1, wherein said antibody or an antigen-bindingfragment thereof binds a protein consisting of (b).
 4. The method ofclaim 1, wherein said antibody or an antigen-binding fragment thereofbinds a protein consisting of (c).
 5. The method of claim 1, whereinsaid antibody or an antigen-binding fragment thereof binds a proteinconsisting of (d).
 6. The method of claim 1, wherein said antibody or anantigen-binding fragment thereof binds a protein consisting of (e). 7.The method of claim 1, wherein said antibody or an antigen-bindingfragment thereof binds a protein consisting of (f).
 8. The method ofclaim 1, wherein said antibody or an antigen-binding fragment thereofbinds a protein consisting of (g).
 9. The method of claim 1, whereinsaid antibody or an antigen-binding fragment thereof binds a proteinconsisting of (h).
 10. The method of claim 1, wherein said antibody oran antigen-binding fragment thereof binds a protein consisting of (i).11. The method of claim 1, wherein said antibody or an antigen-bindingfragment binds a protein consisting of (j).
 12. The method of claim 1,wherein said antibody or an antigen-binding fragment thereof binds aprotein consisting of (k).
 13. The method of claim 1, wherein saidantibody or an antigen-binding fragment thereof binds a proteinconsisting of (l).
 14. The method of claim 1, wherein said antibody oran antigen-binding fragment thereof binds a protein consisting of (m).15. The method of claim 1, wherein said antibody or an antigen-bindingfragment thereof binds a protein consisting of (n).
 16. The method ofclaim 1, wherein said antibody or an antigen-binding fragment thereofbinds a protein consisting of (o).
 17. The method of claim 1, whereinsaid antibody or an antigen-binding fragment thereof binds a proteinconsisting of (p).
 18. The method of claim 1, wherein said antibody oran antigen-binding fragment thereof binds a protein consisting of (q).19. The method of claim 1, wherein said subject is administered anantibody.
 20. The method of claim 1, wherein said subject isadministered an antigen-binding fragment.
 21. The method of claim 1,wherein said immune response is a pro-inflammatory immune response. 22.The method of claim 1, wherein the isolated antibody or anantigen-binding fragment thereof is humanized.
 23. The method of claim1, wherein said protein is glycosylated.
 24. The method of claim 1,wherein said subject is human.